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Carbohydrate utilization in selected strains of British Columbia chinook salmon Mazur, Carol Nelson 1990

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CARBOHYDRATE UTILIZATION IN SELECTED STRAINS OF BRITISH COLUMBIA CHINOOK SALMON by C a r o l Nelson Mazur B.Sc.,  McGill University,  1986  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department  of Animal Science)  We accept t h i s t h e s i s as to the  conforming  r e q u i r e d standard  THE UNIVERSITY OF BRITISH COLUMBIA September ©  1990  C a r o l Nelson Mazur,  1990  In  presenting  degree freely  at  the  available  copying  of  department publication  this  of  in  partial  fulfilment  of  the  University  of  British  Columbia,  I  agree  for  this or  thesis  reference  thesis by  this  for  his thesis  and  study.  scholarly  or for  her  representatives.  financial  of  ANIMAL  SCIENCE  The University of British Columbia Vancouver, Canada  Date  DE-6  (2/88)  September  25, 1990.  gain  shall  that  agree  purposes may  permission.  Department  I further  requirements  be  It not  that  the  Library  by  understood be  an  advanced  shall  permission for  granted  is  for  allowed  the that  without  make  it  extensive  head  of  copying my  my or  written  ii Abstract Digestible chinook salmon diets,  carbohydrate i s commonly encountered by {Oncorhynchus  although l i t t l e  is  tshawytscha)  known r e g a r d i n g i t s  T h i s study was undertaken to examine carbohydrate d i e t of  and (2)  (1)  days.  strains  were fed  a high or a low carbohydrate d i e t  The d i e t s were i s o n i t r o g e n o u s ,  respectively  in chinook  30 % g e l a t i n i z e d  amount of h e r r i n g o i l .  initially  period.  to  for 63  and contained  wheat s t a r c h or an e q u i c a l o r i c  There was an o v e r a l l r e d u c t i o n in  growth of chinook fed the h i g h - c a r b o h y d r a t e d i e t day feeding  salmon  strains.  Y e a r l i n g chinook salmon of three either  utilization.  the e f f e c t s of a h i g h  glucose t o l e r a n c e  s e l e c t e d B r i t i s h Columbia  satiation  in p r a c t i c a l c u l t u r e  Although s p e c i f i c  over the  growth r a t e s  in the high c a r b o h y d r a t e - f e d groups,  63-  declined  they were  comparable to those of c o n t r o l groups in the f i n a l t h i r d of the t r i a l ,  i n d i c a t i n g an a d a p t a t i o n  high carbohydrate d i e t and decreased  carcass  intake was g e n e r a l l y feeding  response.  had i n c r e a s e d c a r c a s s fat  levels relative  lower  the  p r o t e i n and a s h ,  to c o n t r o l s .  in these groups,  Feed  and d i f f e r e n c e s  in  response were observed between d i e t s and s t r a i n s .  Although feed and energy e f f i c i e n c i e s fed the high carbohydrate d i e t , comparable on the two d i e t s , effect  Chinook fed  of  were reduced in chinook  protein u t i l i z a t i o n  was  indicating a protein-sparing  the c a r b o h y d r a t e .  Consumption of the high carbohydrate d i e t significant  e l e v a t i o n s in hepatosomatic  indices  led  to  (HSI) and  iii  l i v e r glycogen  (LG) c o n c e n t r a t i o n s .  l e v e l s exceeding  10 % d i d not appear to have any d e t r i m e n t a l  e f f e c t s on f e e d i n g , HSI f e l l  growth or h e a l t h .  LG c o n c e n t r a t i o n s and  to b a s a l l e v e l s i n a l l groups 21 days a f t e r  withdrawal. example,  In Quesnel chinook, LG  Some s t r a i n d i f f e r e n c e s  were e v i d e n t .  feed For  B i g Qualicum chinook fed the high carbohydrate d i e t  e x h i b i t e d the lowest l i v e r glycogen a c c u m u l a t i o n , highest of c a r c a s s  fat d e p o s i t i o n ,  and best energy e f f i c i e n c y  r e l a t i v e to c o n t r o l groups,  suggesting  carbohydrate metabolism i n t h i s  strain.  Quesnel chinook e x h i b i t e d the highest high carbohydrate d i e t . diet  a difference  Mortality,  rate  ratios  in  On the other hand,  r e l a t i v e growth on the  although unaffected by  i n the Quesnel and Robertson Creek chinook, appeared to  be higher i n high c a r b o h y d r a t e - f e d B i g Qualicum chinook. In the second p a r t of the study, to an o r a l glucose persistent  Strain differences  response.  testing  subjected  t e s t d i s p l a y e d pronounced and  hyperglycaemia, i n d i c a t i v e of poor  tolerance. of  tolerance  chinook salmon  were evident  glucose  i n the magnitude  A c c l i m a t i o n to a h i g h carbohydrate d i e t p r i o r  r e s u l t e d in a s i g n i f i c a n t l y  reduced  elevation  of  blood g l u c o s e ,  i n d i c a t i n g an a d a p t a t i o n response.  plasma glucose  c o n c e n t r a t i o n s approached 500 mg/dl i n some  trials, rise,  plasma i n s u l i n c o n c e n t r a t i o n s  with i n d i s t i n c t peaks.  While  exhibited a two-fold  Plasma glucose  and plasma  i n s u l i n c o n c e n t r a t i o n s were p o o r l y c o r r e l a t e d , glucose  to  i n d i c a t i n g that  i s a poor i n s u l i n secretagogue i n chinook salmon.  iv  TABLE OF  CONTENTS  ABSTRACT TABLE OF  1  i i CONTENTS  L I S T OF  TABLES  L I S T OF  FIGURES  iv vii ix  ACKNOWLEDGEMENTS  x  INTRODUCTION  1  2 2.1  LITERATURE REVIEW Carbohydrate i n Salmonid D i e t s : L i t e r a t u r e Recommendations and C o n t r o v e r s y 2.2 Energetics 2.3 Digestibility 2.4 Carbohydrate Energy Values 2.5 Temperature E f f e c t s 2.6 Fish Size 2.7 Carbohydrate Source 2.8 I n d i g e s t i b l e C a r b o h y d r a t e ( E f f e c t s of F i b e r ) 2.9 S p e c i e s Comparisons ( F i s h ) 2.9.1 S t r a i n Comparisons . 2.10 Carbohydrate Structure 2.11 C a r b o h y d r a t e D i g e s t i o n and A b s o r p t i o n 2.12 Carbohydrate Metabolism 2.12.1 G l y c o l y s i s and G l u c o n e o g e n e s i s 2.12.1.1 Enzyme A d a p t a t i o n 2.12.2 G l y c o g e n S y n t h e s i s and Breakdown 2.12.2.1 Liver glycogen Studies 2.13 Endocrine Control 2.13.1 Insulin 2.13.2 Glucagon 2.13.3 T h y r o i d Hormone 2.14 Oral Glucose Tolerance  4 4 6 7 9 10 1 1 12 14 16 17 19 19 20 21 25 26 30 31 31 35 35 35  3 3.1  GENERAL MATERIALS AND METHODS H i s t o r y and M a i n t e n a n c e o f E x p e r i m e n t a l F i s h  39 39  4  EXPERIMENT 1. The E f f e c t s of F e e d i n g a H i g h V e r s u s Low C a r b o h y d r a t e D i e t on Growth, Body C o m p o s i t i o n , F e e d and P r o t e i n U t i l i z a t i o n , L i v e r G l y c o g e n C o n c e n t r a t i o n and L i v e r W e i g h t i n S e l e c t e d S t r a i n s of B. C. C h i n o o k Salmon.  42  V  4.1 MATERIALS AND METHODS D i e t P r e p a r a t i o n and Composition 4. 1 . 2 4. 1 . 3 F i s h S e l e c t i o n and Tank A l l o c a t i o n S t a r t of Experiment 4. 1 .4 4. 1 . 5 Proximate Body Composition 4. 1 . 6 L i v e r Glycogen Determination 4. 1 .7 S t a t i s t i c a l Analyses  42 42 45 46 47 49 50  4. 2 RESULTS 4. 2. 1 Proximate Analyses of D i e t s and P r o t e i n Sources Growth and C o n d i t i o n F a c t o r 4. 2. 2 4. 2. 3 Carcass Composition 4. 2. 3 . 1 Growth in Terms of P r o t e i n and Fat Feed Intake and E f f i c i e n c y I n d i c e s 4. 2. 4 4. 2. 5 Protein U t i l i z a t i o n 4. 2. 6 L i v e r Glycogen and Hepatosomatic Index 4. 2. 7 Mortality  51 51  4. 4. 4. 4. 4. 4. 4. 4. 4. 4.  3 3. 3. 3. 3. 3. 3. 3. 3. 3.  DISCUSSION 1 General 2 Growth Rates 3 Carcass Composition 3.1 Growth in Terms of Carcass Composition 4 Feed Intake and Feeding Response 5 Feed and Energy E f f i c i e n c y 6 U t i l i z a t i o n of P r o t e i n 7 Liver Effects 8 Mortality  4. 4  CONCLUSIONS  5  EXPERIMENT 2  5. 1 5. 5. 5. 5. 5. 5. 5. 5. 5.  1 .1 1 .1 .2 1 . 1 .3 1 . 1 .4 1 . 1 .5 1 .1 .6 1 . 1 .7 1 . 1 .8 1 .1 .9  Part  i)  (Experiment  1)  Oral Glucose Tolerance i n S e l e c t e d S t r a i n s of B. C . Chinook Salmon.  MATERIALS AND METHODS Tank P r e p a r a t i o n and F i s h A l l o c a t i o n Glucose A d m i n i s t r a t i o n : Preliminary Glucose Tolerance T r i a l s Experimental Sampling Blood Sampling Technique Plasma Glucose Determination Plasma I n s u l i n Radioimmunoassay S t a t i s t i c a l Analysis  51 60 64 64 70 73 76 78 78 79 80 80 81 82 85 86 87 89  92 92 92 92 Trial 93 96 97 97 98 99 100  vi  5.2  Part i i )  5.2.1 5.2.1.1 5.2.1.2 5.3 5.3.1 5.3.1.1 5.3.1.2 5.3.2 5.3.2.1 5.3.2.2  O r a l Glucose Tolerance i n Chinook Salmon A c c l i m a t e d to High and Low Carbohydrate D i e t s R e s p e c t i v e l y  MATERIALS AND METHODS Glucose Tolerance T r i a l s S t a t i s t i c a l Analysis RESULTS Part i ) O r a l Glucose Tolerance in Chinook Salmon of S e l e c t e d S t r a i n s Plasma Glucose Response Plasma I n s u l i n Response Part i i ) E f f e c t of P r e - T e s t D i e t on O r a l Glucose T o l e r a n c e Plasma Glucose Response Plasma I n s u l i n response  101 101 101 102 103 103 103 106 107 110 110  5.4 DISCUSSION 5.4.1 O r a l Glucose T o l e r a n c e : General 5.4.1.1 Part i ) O r a l Glucose Tolerance i n Chinook Salmon of S e l e c t e d S t r a i n s 5.4.2.1 Part i i ) E f f e c t of P r e - T e s t D i e t on O r a l Glucose Tolerance  117  5.5  119  6  CONCLUSIONS  REFERENCES  (Experiment 2)  112 112 114  121  vii  LIST OF TABLES Table  1 - Composition of t e s t d i e t s as f e d .  43  Table 2 - C a l c u l a t e d m e t a b o l i z a b l e energy values of t e s t d i e t s on a m o i s t u r e - f r e e b a s i s . Table 3 - Proximate analyses  of d i e t s .  Table 4 - Proximate analyses of p r o t e i n sources for experimental d i e t s (dry matter b a s i s ) . Table 5 - Mean body weights of treatment groups at 21day i n t e r v a l s d u r i n g the experiment.  44 52 53 55  Table 6 - Average body weight gains (BWG) and percent BWG of treatment groups at the end of the 63-day feeding p e r i o d .  57  Table 7 - S p e c i f i c growth r a t e s of treatment groups.  58  Table 8 - Mean c o n d i t i o n f a c t o r s i n treatment groups, i n i t i a l l y and at the end of the 63-day feeding p e r i o d .  61  Table 9 - Proximate analyses  62  Table  Table  Table  Table  Table  of whole f i s h .  10 - Instantaneous p r o t e i n gain (IPG) and instantaneous l i p i d gain (ILG) in treatment groups over the 63-day feeding p e r i o d .  65  11 - Mean p r o t e i n weights of chinook i n treatment groups, i n i t i a l l y and at the end of the 63-day feeding t r i a l .  66  12 - Feed intake ( F I ) , feed e f f i c i e n c y and energy e f f i c i e n c y (EE) r a t i o s i n the treatment groups at the end of the 63-day feeding p e r i o d .  67  13 - P r o t e i n e f f i c i e n c y r a t i o s (PER) and p r o d u c t i v e p r o t e i n values (PPV) i n treatment groups over the 63-day feeding p e r i o d .  71  14 - Mean percent l i v e r glycogen c o n c e n t r a t i o n s and hepatosomatic i n d i c e s (HSI) i n treatment groups ( i n i t i a l l y , d u r i n g feeding and 21 days post-feed withdrawal.  74  Table 15 - T o t a l m o r t a l i t i e s i n treatment groups over the 63-day feeding p e r i o d .  77  viii  Table 16 - Mean plasma glucose and i n s u l i n concent r a t i o n s i n 3 s t r a i n s of chinook salmon subjected to an o r a l glucose t o l e r a n c e t e s t (GTT). Table  104  17 - Mean plasma glucose and i n s u l i n concent r a t i o n s i n C a p i l a n o chinook salmon s u b j e c t e d to an o r a l glucose t o l e r a n c e t e s t (GTT) a f t e r feeding low and high carbohydrate p r e - t e s t diets respectively. 108  ix  LIST OF FIGURES Figure  1 - Glucose Metabolism : g l y c o l y s i s and gluconeogenesis pathways.  22  F i g u r e 2 - L i v e r Glycogen S y n t h e s i s and Breakdown.  27  F i g u r e 3 - Growth of Chinook Salmon of Three S t r a i n s Fed Low VS High Carbohydrate D i e t s over a 63-Day P e r i o d .  54  F i g u r e 4 - Twenty-One Day I n t e r v a l S p e c i f i c Growth Rates of Chinook Salmon of Three S t r a i n s Fed Low VS High Carbohydrate D i e t s .  59  F i g u r e 5 - E f f e c t of D i e t a r y Carbohydrate on Body Composition of Chinook Salmon of Three S t r a i n s Fed Low or High Carbohydrate D i e t s for 63 Days.  63  F i g u r e 6 - P r o t e i n Weights of Chinook Salmon Fed Low or High Carbohydrate D i e t s for 63 Days.  68  F i g u r e 7 - Feed E f f i c i e n c y and Energy E f f i c i e n c y R a t i o s of Chinook Salmon of Three S t r a i n s Fed Low or High Carbohydrate D i e t s for 63 Days.  69  F i g u r e 8 - P r o t e i n E f f i c i e n c y R a t i o s and P r o d u c t i v e P r o t e i n Values of Chinook Salmon of Three S t r a i n s Fed Low or High Carbohydrate D i e t s for 63 Days.  72  F i g u r e 9 - E f f e c t of D i e t a r y Carbohydrate on L i v e r Glycogen L e v e l in Chinook Salmon of Three Strains: During Feeding and Subsequent to Feed Withdrawal.  75  Figure Figure Figure  10 - P r e l i m i n a r y Comparison T r i a l of Glucose A d m i n i s t r a t i o n : Capsules VS S o l u t i o n .  95  11 - O r a l Glucose Tolerance Test Response in Chinook Salmon of S e l e c t e d B. C . S t r a i n s .  105  12 - E f f e c t of P r e - T e s t D i e t on O r a l Glucose Tolerance i n C a p i l a n o Chinook Salmon.  109  X  ACKNOWLEDGEMENTS T h i s research was supported through a Department of F i s h e r i e s and Oceans grant to D r . D. A . H i g g s . I would l i k e to thank D r . B . E . March and D r . D. A . Higgs for the continued support, guidance and commitment they have shown throughout t h i s p r o j e c t . I would a l s o l i k e to thank Bakhshish Dosanjh for h i s valued guidance i n laboratory analyses. The generous a s s i s t a n c e of D r . E . P l i s e t s k a y a in q u a n t i f y i n g the plasma i n s u l i n v a l u e s for t h i s research is greatly appreciated. I would l i k e to thank B r i a n Toy for h i s i n v a l u a b l e a s s i s t a n c e in many aspects of t h i s study. I would a l s o l i k e to thank members of the f a c u l t y and s t a f f and students at U . B . C . for t h e i r a s s i s t a n c e , and for making my s t u d i e s most enjoyable. F i n a l l y , I would l i k e to thank my husband C a r l for h i s l o v e , encouragement and sense of humour throughout the project.  1  1  INTRODUCTION Salmonids  are  a r e among t h e 85  carnivores,  carbohydrate.  and The  as a p e r c e n t a g e terrestrial energy  energy  protein  less  for locomotion  protein  from  p r o t e i n s and  use p r o t e i n  energy (Smith  of  their  an  energetically  uric  per  Ryman, key  expressed  fish,  when  that  of  protein  energy  (Tytler  and  to  total  Calow,  1985).  require  T h i s i s because  less  the  of the environment,  (Cowey,  1980)  energy  and  1978;  can  requires  effectively  f o r s y n t h e s i s of  source.  In f a c t ,  over c a r b o h y d r a t e  Cowey and  inexpensive process  unit  Luquet,  exhibit  i n g e s t e d than a number of  dietary  t o be d i a b e t i c ,  b l o o d g l u c o s e and 1972).  ability  fish other  energy  as  indicated  poor  insulin  Moreover, they possess  in  their  status low  adaptation  or  more  their generally  inability  (Palmer  activity  i n carbohydrate metabolism,  f o r enzymatic  Most  animals.  They a r e by  for  gills;  to urea  to retain  limitations  carbohydrate.  fish  1983).  i n comparison  T h i s a l l o w s the  protein  enzymes i n v o l v e d  limited  that  as an  et-al.,  to u t i l i z e  considered control  of  perspective, fish  preferentially  production.  Salmonids ability  little  waste n i t r o g e n i s e x c r e t e d a s ammonia v i a t h e  acid  energy  contains very  b o t h a s a s o u r c e o f amino a c i d s  body t i s s u e  metabolism  species that  does n o t have t o m a i n t a i n a body  different  use  1 : 2  warm-blooded a n i m a l s .  temperature energy  dietary  from a n o t h e r  fish  diet  i s 2 to 4 times  i s approximately  poikilothermic  known f i s h  requirement  of the d i e t ,  at t h i s than  natural  homeotherms and  ratio  Looking  their  % of  (Hilton  to  and  levels  of  and  display  and  Slinger,  a  2  1982).  Consumption of high d i e t a r y  l e v e l s of  carbohydrate leads to e x c e s s i v e l i v e r glycogen ( H i l t o n and A t k i n s o n , 1982; 1972;  P h i l l i p s et  al.,  Bergot,  digestible accumulation  1979a; Palmer and Ryman,  1948).  In f o r m u l a t i n g p r a c t i c a l d i e t s for c u l t u r e d carbohydrate  is  important for s e v e r a l  reasons.  source of d i e t a r y energy for use  in s p a r i n g expensive  Also,  i s a major c o n s t i t u e n t of  carbohydrate  and pressure biological filler  Diet processing  form of  and  fish  contains  considerable  species.  at.,  1986;  Buhler and H a l v e r ,  These  Hilton  1961;  The determination  its  its  pellet  controversy  et  include:  al.,  P h i l l i p s et  of  ( H i l t o n et  al.  1982; al . ,  fact  salmonids  range from  level  of an optimal l e v e l  i s hindered by the  digestion,  for  Research recommendations  d i g e s t i b l e carbohydrate for salmonids  affect  heat  Carbohydrate i s often used as a  9% to 30% as the maximum a c c e p t a b l e d i e t a r y  carbohydrate  fish  s t a r c h and increase  r e g a r d i n g the optimal d i e t a r y carbohydrate l e v e l  Beamish et  sources  integrity.  The l i t e r a t u r e  and other  starch  expensive marine  i n d i e t s and a l s o as a binder to give good  stability  dietary  methods which i n v o l v e  p a r t i a l l y h y d r o l y z e the  availability.  in the  expensive  inexpensive p l a n t p r o t e i n  which are employed to p a r t i a l l y r e p l a c e protein sources.  least  instance,  it  for growth.  the  For  on a c o s t per u n i t weight b a s i s ,  protein  is  salmonids,  Bergot,  1987a;  1979a;  1948). of  dietary  that so many  metabolism and u t i l i z a t i o n  variables by the  carbohydrate source and form, d i e t a r y  fish.  level,  3  processing  of the d i e t  (ie.  steam versus  water temperature and s a l i n i t y , s u b s p e c i e s , age,  life  season,  stage and s i z e of  and d u r a t i o n on a p a r t i c u l a r d i e t , environmental and d i e t a r y  extrusion  pelleting),  f i s h s p e c i e s and fish,  stress,  feeding  and other  factors.  The n u t r i t i o n a l needs of post j u v e n i l e  chinook  in  water are v i r t u a l l y unknown even though t h i s s p e c i e s presently  p r e f e r r e d for c u l t u r e  formulations  is  in B r i t i s h Columbia.  (Salmo  salar).  (Oneorhynchits  mykiss)  Diet  and A t l a n t i c  In a d d i t i o n , a wide range of  carbohydrates has been t e s t e d in salmonid d i e t s : sugars  such as g l u c o s e , which i s  carbohydrates,  especially  in t h e i r d i g e s t i o n  from simple  r e a d i l y absorbed,  starches,  and a b s o r p t i o n .  to  which are h i g h l y  exhibit  1961).  The l a t t e r  Carbohydrate u t i l i z a t i o n  authors r e p o r t e d that  a marked a b i l i t y to u t i l i z e  s u p e r i o r u t i l i z a t i o n of  of high versus  lower molecular weight  and l i v e r glycogen  s e l e c t e d B . C . stocks h e l d i n sea water,  carbohydrates. (1)  the  (a)  feed  in chinook of  and (2)  oral  as an index of carbohydrate u t i l i z a t i o n  a c c l i m a t e d chinook of  chinook  low carbohydrate d i e t s on growth,  and p r o t e i n u t i l i z a t i o n ,  tolerance  (Buhler  d i e t a r y c a r b o h y d r a t e , and  T h i s study was undertaken in order to examine: effects  complex variable  in chinook salmon has been examined in only one study and H a l v e r ,  sea  c u r r e n t l y in use have been d e r i v e d p r i m a r i l y from  research on rainbow t r o u t salmon  level  s e l e c t e d B . C . stocks and  glucose  in sea  water  (b)  s e l e c t e d stocks p r e - a c c l i m a t e d to high and low carbohydrate diets,  respectively.  4  2 2.1  LITERATURE REVIEW Carbohydrate i n Salmonid D i e t s : L i t e r a t u r e Recommendations and Controversy There i s c o n s i d e r a b l e debate  i n the  regarding the optimum d i e t a r y l e v e l carbohydrate for salmonids. conducted on d i f f e r e n t efficiency,  digestible  Numerous s t u d i e s have been  f i s h s p e c i e s examining growth,  digestibility,  tolerance,  of  literature  enzyme a d a p t a t i o n ,  feed  glucose  metabolism and other parameters i n order to  assess carbohydrate u t i l i z a t i o n . Hilton  et  al.  (1982) made a d i s t i n c t i o n between maximum  tolerable digestible digestible  carbohydrate and optimum l e v e l of  carbohydrate.  The former term r e f e r s to  " l e v e l which can be e f f e c t i v e l y ...without  d i g e s t e d and metabolized  causing growth impairment or i n c r e a s e  m o r t a l i t y rate" and the l a t t e r be e f f i c i e n t l y  digested  u t i l i z e d as an energy Hilton effectively  et  al.  "the l e v e l  in  that cannot only  and metabolized but a l s o  efficiently  source".  (1987a) concluded that  salmonids cannot  u t i l i z e more than 14% (140 g/kg)  carbohydrate in the d i e t , rainbow t r o u t .  the  Above t h i s  digestible  based on feeding t r i a l s with level  no f u r t h e r a d a p t a t i o n of  key carbohydrate c a t a b o l i z i n g enzymes o c c u r r e d ( H i l t o n and Atkinson,  1982).  growth depression  In 1987,  H i l t o n demonstrated  i n rainbow t r o u t fed a 25% glucose  with 43% p r o t e i n as compared to an isoenergetic  significant  control diet  ( H i l t o n et  diet  isonitrogenous al . ,  1987b).  Phillips  5  et  al.  (1948) found that young brook t r o u t  fontinalis),  s u p p l i e d with more than 9% d i g e s t i b l e  carbohydrate in t h e i r d i e t , high-glycogen  e x h i b i t e d growth r e d u c t i o n ,  l i v e r s and i n c r e a s e d  Reports by other i n d i c a t i n g that  mortality.  investigators  20% (200  g/kg)  carbohydrate i s e f f e c t i v e l y  have been c o n t r a d i c t o r y ,  or more of  utilized.  and Halver (1961) r e p o r t e d that of  (Salvelinus  digestible  For example,  up to 48% of d e x t r i n or 20%  low molecular weight sugars c o u l d be t o l e r a t e d  salmon  (Oncorhynchus  tshawytscha)  rainbow t r o u t  by chinook  with no p a t h o l o g i c a l  consequences or growth d e p r e s s i o n . noted that  Buhler  Moreover, Luquet (1971)  fed a 30% p r o t e i n 50% raw corn  starch diet  e x h i b i t e d the  utilization  than those fed d i e t s c o n t a i n i n g twice as much  protein.  Further,  same growth as,  Bergot  and b e t t e r  (1979a) r e p o r t e d  protein  effective  u t i l i z a t i o n of a 30% glucose d i e t  by rainbow t r o u t when  d i e t a r y p r o t e i n content was 45%.  However, when the  level  was lowered to 30%, growth depression  Similarly,  Luquet et  al.  35%.  was decreased  In a 1986 study by Beamish et  30% glucose d i e t  resulted.  (1975) found a d e c l i n e  u t i l i z a t i o n when p r o t e i n l e v e l  had s i g n i f i c a n t l y  carcass  fat,  but s i g n i f i c a n t l y  carcass  p r o t e i n than t r o u t  al.,  protein  in sucrose  from 55% to  rainbow t r o u t  fed a  lower body weights and  higher feed c o n v e r s i o n and  fed an i s o n i t r o g e n o u s  diet  with  18% l i p i d . Based on evidence i n the  literature,  (1979) s t a t e d that carbohydrate  Cowey & Sargent  ( d e x t r i n or s t a r c h )  levels  6  of approximately 25% of the d i e t e q u i c a l o r i c amounts of  fat  are as e f f e c t i v e  for energy  s p e c i e s i n c l u d i n g channel c a t f i s h , (NRC,  1981).  Hilton,  "tolerable" level  however,  for a v a r i e t y of  the maximum  carbohydrate for  salmonids  i s approximately 200 g/kg d i e t ,  while the  in the neighborhood of  ( H i l t o n and A t k i n s o n ,  Disagreement  140 g/kg  in the l i t e r a t u r e  many f a c t o r s complicate carbohydrate l e v e l . to e x t r a p o l a t e another.  Also,  digestibilities  is  1982).  the d e t e r m i n a t i o n of an i d e a l  Species d i f f e r e n c e s  experimental r e s u l t s the compositions  make i t  difficult  from one s p e c i e s  to  of the experimental  of the t e s t carbohydrate s o u r c e s ,  diets,  and water  studies.  Energetics it  of  "optimal" l e v e l  i s not s u r p r i s i n g , as  temperatures have v a r i e d widely between 2.2  fish  rainbow t r o u t and p l a i c e  recommended that  of d i g e s t i b l e  as  is essential  to understand the energy  requirements  salmonids to minimize the use of d i e t a r y p r o t e i n for  energy by these s p e c i e s .  The p r o t e i n - s p a r i n g a c t i o n of  l i p i d s has been confirmed i n s e v e r a l Kaushik,  1985;  Beamish and Medland,  value of d i g e s t i b l e (Kaushik et  al.,  carbohydrate i s  1989).  f i s h species  (Cho and  1986), but the  energy  still  in q u e s t i o n  Many s t u d i e s i n d i c a t e  have a l i m i t e d a b i l i t y to adapt to increased carbohydrate  (Cowey et  F u r u i c h i and Yone, can spare p r o t e i n  al . ,  1980)  1977a; Cowey et  and that  that  fish  dietary al.,  1977b;  i n c r e a s i n g carbohydrate  (Pieper and P f e f f e r ,  1979;  Shimeno et  al.,  7  1979,  1985).  F i s h are s a i d to eat  needs (Kaushik and Luquet, 1984). protein:energy  energy depends,  Therefore the  d i e t a r y p r o t e i n content  (Bergot,  al.,  1979a).  dietary the  1982)  and the  H i l t o n et  i s probably not  al. glucose being  energy.  Digestibility Carbohydrate d i g e s t i b i l i t y  can be a major o b s t a c l e  determining the a p p r o p r i a t e d i e t a r y carbohydrate l e v e l salmonids.  The d i g e s t i b i l i t y  many f a c t o r s , level  of  Slinger,  i n c o r p o r a t i o n in the d i e t ,  1983;  fecal  processing  collection  Spannhof and P l a n t i k o w ,  Bergot and Breque,  monosaccharide  technique,  ( H i l t o n and  1983;  Takeuchi el  its  digestibility.  1967;  Bergot,  the  The  absorbed.  By c o n t r a s t ,  whereas complex h i g h l y branching carbohydrates are not  Nose,  al.  1983).  glucose i s completely  d i g e s t e d by most f i s h ,  for  source,  The degree of p o l y m e r i z a t i o n of a carbohydrate i s main element a f f e c t i n g  in  of carbohydrate depends on  i n c l u d i n g carbohydrate s t r u c t u r e ,  f i s h s p e c i e s and method of  1979;  for  alternative  salmonids may ' t o l e r a t e '  l e v e l s in excess of 25%, the sugar used for  l e v e l s of  ( H i l t o n et  (1982) suggested that while  2.3  energy  The u t i l i z a t i o n of glucose  in l a r g e p a r t , on the  sources in the d i e t  efficiently  their  (P/E) r a t i o i s c r i t i c a l for maximizing  use of p r o t e i n for growth.  energy  to s a t i s f y  i n c l u d i n g rainbow t r o u t  well  (Singh and  1979a), which are poor s t a r c h d i g e s t e r s  (Spannhof and P l a n t i k o w ,  1983).  Studies  indicate  that  the  8  digestibility 1967;  of  raw s t a r c h i s below 50% ( P h i l l i p s et  Bergot and Breque,  1983).  Carbohydrate d i g e s t i b i l i t y increasing dietary l e v e l , can a f f e c t  a l s o decreases with  and the amount of  starch in a diet  the a b s o r p t i o n of other carbohydrates i n c l u d i n g  simple sugars.  While the d i g e s t i b i l i t y  r e l a t i v e l y unaffected al . ,  by i t s  level  1982), the d i g e s t i b i l i t i e s  of glucose  i n the d i e t  c o r r e l a t e d with d i e t a r y l e v e l al.  ( H i l t o n et  negatively  (Singh and Nose,  (1963) found the d i g e s t i b i l i t y  1967).  in rainbow t r o u t  Inaba  of cooked wheat a -  s t a r c h to be 90% and 48.2% at d i e t a r y l e v e l s of 40%, r e s p e c t i v e l y ,  is  of more complex  carbohydrates such as s t a r c h and d e x t r i n are  et  al.,  11.5% and  (from Singh and Nose,  1967). When raw s t a r c h i s exposed processing  i t becomes g e l a t i n i z e d ,  G e l a t i n i z a t i o n of s t a r c h g r e a t l y (Bergot and Breque, source  1980a,  reflected  1980b).  by e l e v a t e d  hepatosomatic  indices  protein efficiency  making i t s  digestibility  efficiency  as an energy  (HSI),  ratios  (Pieper and  This increased a v a i l a b i l i t y  blood glucose l e v e l s ,  is  increased  improved feed c o n v e r s i o n and  (Kaushik and de O l i v a  Teles,  of g e l a t i n i z e d corn s t a r c h at a d i e t a r y  30% i n rainbow t r o u t .  trout  improves i t s  Bergot and Breque (1983) reported a 90%  digestibility of  1983)  or p a r t i a l l y h y d r o l y z e d .  for t r o u t comparable to l i p i d s or p r o t e i n  Pfeffer,  1985).  to heat and p r e s s u r e d u r i n g  Other i n v e s t i g a t i o n s  i n d i c a t e marked improvements  level  on rainbow  in d i g e s t i b i l i t y  and  9  energy a v a i l a b i l i t y of s t a r c h by e x t r u s i o n p r o c e s s i n g or cooking  (Kaushik and O l i v a T e l e s ,  Carbohydrate d i g e s t i b i l i t y different that  s p e c i e s of f i s h .  the d i g e s t i b i l i t i e s  and s t a r c h  (Shimeno et  1985;  Inaba et  al.,  1963).  v a r i e s g r e a t l y among  It has been demonstrated i n c a r p  of d e x t r i n al.,  1979;  (Takeuchi et  al .  Chiou and Ogino,  d i e t a r y l e v e l s ranging from 10 to 40% are f a i r l y  1979)  1975)  s t a b l e and  much higher than those found in c a r v i v o r o u s s p e c i e s . contrast, yellowtail and Nose,  digestibility (Shimeno et 1967;  2.4  In  of s t a r c h by c a r n i v o r e s such as al . ,  Inaba et  1979)  al.,  and rainbow t r o u t  1963)  with i n c r e a s i n g d i e t a r y l e v e l , digestibility  at  significantly  and g r e a t l y  the  (Singh  declines  reduces  the  of d i e t a r y p r o t e i n .  Carbohydrate Energy Values Caution must be e x e r c i s e d  carbohydrates i n f i s h d i e t s  in a s s i g n i n g energy values  (Jobling,  1983).  H i l t o n et  (1987a) recommended that  the use of d i g e s t i b l e  and m e t a b o l i z a b l e  (ME) v a l u e s ,  overestimate  energy  p r o d u c t i v e energy v a l u e s ,  which tend  percentage  These i n v e s t i g a t o r s  which i s  of  the  is  c a l c u l a t e d NE  for glucose and s t a r c h i n rainbow t r o u t ,  a dietary level  for  They proposed  of gross energy of a carbohydrate that  r e t a i n e d i n the c a r c a s s . values  to  be d i s c o n t i n u e d  (NE) value as an a l t e r n a t i v e ,  al .  energy (DE)  determining u t i l i z a b l e energy of t r o u t d i e t s . the net energy  to  each fed at  25%, to be approximately 25% of  glucose gross energy and 13% of the s t a r c h gross  the  energy.  10  These v a l u e s especially  were c o n s i d e r a b l y lower than  expected,  for glucose which i s almost completely  Although NE values may be more t e n a b l e , and can be a l t e r e d by many f a c t o r s  absorbed.  they are not  that a f f e c t  fixed  carbohydrate  digestibility. One cannot c o n s i d e r only the c a l c u l a t e d energy of c a r b o h y d r a t e s . effect  diet,  Carbohydrates have been shown to have an  on v o l u n t a r y feed  starch,  with i t s  intake.  Studies  low d i g e s t i b i l i t y ,  i n c r e a s i n g food intake  Kaushik and de O l i v a T e l e s ,  show that raw  a c t s as bulk i n the  ( H i l t o n and S l i n g e r . , 1985).  a reduced feed  which c o n t r i b u t e s  to t h e i r growth d e p r e s s i o n  Slinger,  Poor glucose t o l e r a n c e  hyperglycaemia may a f f e c t 2.5  1983;  On the other hand,  fed d i e t s c o n t a i n i n g glucose e x h i b i t  1987a).  values  fish  intake  ( H i l t o n and  and prolonged  appetite.  Temperature E f f e c t s As environmental temperature r i s e s or f a l l s ,  metabolism r e s p e c t i v e l y  increases  u t i l i z a t i o n of energy changes.  the  or d e c r e a s e s , and  Metabolic rate  not  however,  occurs,  in responses between  the d i f f e r e n c e  temperature extremes (Hochachka and Hayes,  as  is  d i r e c t l y r e l a t e d to temperature, lessening  fish's  adaptation  1961).  Routes of energy metabolism have a l s o been shown to change with temperature. 1  ^C l a b e l l e d glucose,  Hochachka and Hayes (1961),  using  demonstrated that d u r i n g c o l d  a c c l i m a t i o n i n brook t r o u t ,  the pentose phosphate pathway  is  11  a c t i v a t e d and fat  s y n t h e s i s i s enhanced.  temperatures, more glucose Meyerhof  (or g l y c o l y s i s )  passes through the Embden-  pathway.  (G6PDH)  phosphate pathway)  H i l t o n et  Similarly,  (1982) found a higher a c t i v i t y of glucose dehydrogenase  At higher  al.  6-phosphate  (the enzyme l e a d i n g i n t o the pentose  i n 11°C a c c l i m a t e d rainbow t r o u t  versus  those h e l d at 1 5 ° C . L i v e r glycogen accumulation and l i v e r weight weight et  : body  r a t i o (LBW) i n c r e a s e as temperature decreases ( H i l t o n  al.,  1982; H i l t o n ,  1982;  P h i l l i p s et al.,  1966), and a  longer f a s t i n g p e r i o d i s r e q u i r e d for l i v e r glycogen to at lower temperatures  (Hilton,  1982).  Seibert  fall  (1985)  concluded that gluconeogenesis i s the predominant source of glucose  at lower temperatures,  enhanced at higher  whereas g l y c o g e n o l y s i s  is  temperatures.  Although i t has been shown that the a b s o r p t i o n of glucose et  al.  i s not s i g n i f i c a n t l y a l t e r e d by temperature ( H i l t o n 1982), s t a r c h d i g e s t i o n  temperatures, uptake) 2.6  slower at  reducing the appearance rate  of l i b e r a t e d  Fish  is  lower (and hence  glucose.  Size  The b a s i c r e l a t i o n s h i p between body weight and rate i s d e s c r i b e d by the equation Y = a w ^ . 8 )  metabolic  where Y i s the metabolic a constant (Halver,  f  r a t e , W i s the body weight and a i s  which v a r i e s with f i s h species and temperature  1989).  Thus as weight  increases,  energy  12  requirement  for metabolism decreases in r e l a t i o n to body  weight. T o l e r a n c e to and u t i l i z a t i o n of carbohydrate are r e p o r t e d to fish size  increase,  (Rychly,  and p r o t e i n requirement decrease,  1980;  al . ,  1948).  2.7  Carbohydrate Source  Austreng et al . ,  1977;  The type or source of carbohydrate present diet the  has a major bearing on how a c c e s s i b l e i t s fish.  In a d d i t i o n ,  the p r o f i l e s  There must be a balance between the rate at carbohydrate becomes a v a i l a b l e the c i r c u l a t i o n )  and i t s  It has g e n e r a l l y  fish  energy  is  higher molecular weight carbohydrates  its  i s a delay  l e v e l s of  species.  (ie.  enters  f i s h grow b e t t e r on  (ie.  starches)  It has been  in a b s o r p t i o n of g l u c o s e ,  u t i l i z a t i o n by a l l o w i n g more time  than on  hypothesized  that when high molecular weight carbohydrates are there  ingested,  which i n c r e a s e s  for the a c t i v a t i o n  carbohydrate metabolism enzymes and i n s u l i n l e v e l s to (Pieper and P f e f f e r ,  1980b; F u r u i c h i and Yone,  sugars  Decreased e f f i c i e n c y  relative  to g e l a t i n i z e d  higher d i e t a r y l e v e l s ,  of  rise  1982a).  There are numerous i n v e s t i g a t i o n s which lend support to hypothesis.  to  utilization.  been r e p o r t e d that  lower molecular weight sugars.  in a  which  for metabolism  rate of  P h i l l i p s , et  and a c t i v i t y  carbohydrate d i g e s t i v e enzymes vary among f i s h  with  of u t i l i z a t i o n of  this  simple  corn s t a r c h , p a r t i c u l a r l y at  has been demonstrated  in rainbow  13  trout  (Pieper and P f e f f e r ,  Akiyama, Murai and Nose, fed g e l a t i n i z e d potato exhibited better fed s e v e r a l  1979,  1980b).  In a 1982 study by  chum salmon f r y (Oncorhynchus  s t a r c h at a d i e t a r y l e v e l  feed and p r o t e i n e f f i c i e n c i e s  of 20%  than those  lower molecular weight c a r b o h y d r a t e s ,  glucose and d e x t r i n .  including  Y e l l o w t a i l fed p u r i f i e d d i e t s  c o n t a i n i n g 20% glucose e x h i b i t e d hyperglycaemia, and s i g n i f i c a n t Starch,  reductions  i n growth and feed  glycosuria,  efficiency.  on the other hand, was absorbed more g r a d u a l l y  allowing Yone,  keta)  it  to be p a r t i a l l y u t i l i z e d  1986).  (Furuichi,  A study on channel c a t f i s h  T a i r a and  i n d i c a t e d that  low  molecular weight sugars were not u t i l i z e d for energy,  and  indeed t h e i r d i e t a r y i n c l u s i o n l e v e l  l e d to decreased  growth  while d e x t r i n and corn s t a r c h  served  and p r o t e i n d e p o s i t i o n , as good energy  sources  Yone (1982b) t e s t e d the d e x t r i n and a - s t a r c h feed e f f i c i e n c y  (Wilson and Poe, relative  glucose,  i n carp and red sea bream.  i n c a r p were best with the s t a r c h  growth d i f f e r e n c e s  carbohydrate sources slightly  F u r u i c h i and  u t i l i z a t i o n of  followed by d e x t r i n , then g l u c o s e . no s i g n i f i c a n t  1987).  starch-fed  diet,  Red sea bream e x h i b i t e d between the  used and feed e f f i c i e n c y  higher in the  Growth and  three was only  fish.  S e v e r a l r e p o r t s appear to be c o n t r a d i c t o r y to those c i t e d above.  For i n s t a n c e ,  Buhler and Halver (1961) found  that young chinook salmon fed d i e t s c o n t a i n i n g 20% of different weight,  carbohydrates grew b e t t e r  r e a d i l y absorbed sugars.  on the  Hung et  lower molecular al.  (1989)  14  reported s i m i l a r r e s u l t s (Aci pens er  i n j u v e n i l e white  transmontanus)  sturgeon  fed p u r i f i e d d i e t s  27.2% of v a r i o u s c a r b o h y d r a t e s .  containing  F i s h fed maltose and  glucose d i e t s e x h i b i t e d s u p e r i o r growth to those fed d i e t s c o n t a i n i n g d e x t r i n or s t a r c h . strategy  used in t h i s  The c o n t i n u a l  feeding  study may have a l l e v i a t e d  plasma  glucose a c c u m u l a t i o n , as suggested by the l a c k of hyperglycaemia i n the g l u c o s e Bergot's  1979  investigation,  and m a l t o s e - f e d  fish.  In  rainbow t r o u t fed a d i e t  c o n t a i n i n g 30% of glucose e x h i b i t e d s u p e r i o r growth, c o n v e r s i o n and p r o t e i n e f f i c i e n c y natural starch .2.8  Carbohydrate ( The E f f e c t s  Commercial and experimental  number of  amount of  of F i b e r )  f i s h d i e t s often  T h i s " f i b e r " c o u l d be any  l i g n i n , pectin,  1984).  i s used as a f i l l e r ,  and raw s t a r c h  In experimental  fish  or as a b i n d e r .  commercial or p r a c t i c a l f o r m u l a t i o n s much of the from the  Studies  i n c l u s i o n of p l a n t p r o t e i n  show that  f i b e r can a f f e c t  u t i l i z a t i o n of many d i e t a r y n u t r i e n t s carbohydrate. the in  vitro  For example, intestinal  diets  to make up the bulk of d i e t s when  m a n i p u l a t i n g l e v e l s of other n u t r i e n t s ,  originates  contain a  or complex carbohydrates i n c l u d i n g  hemicellulose,  (Bromley and A d k i n s , fiber  fiber.  indigestible  cellulose,  r a t i o to those fed a  diet.  Indigestible  significant  feed  In  fiber  sources.  the uptake and  including  digestible  f i b e r has been shown to  reduce  a b s o r p t i o n of glucose and the  15  i n T i l a p i a (Oreochromis  u t i l i z a t i o n of d i e t a r y d e x t r i n (Shiau  et  al . ,  remain p o o r l y  1989).  sp.)  The p h y s i o l o g i c a l e f f e c t s of f i b e r  understood i n f i s h .  F i b e r a l s o a l t e r s the r a t e of g a s t r i c e v a c u a t i o n . While f i b e r sources such as c a r b o x y m e t h y l c e l l u l o s e or c e l l u l o s e have been shown t o increase emptying 1983)  (Shiau  et  the speed of stomach  al ., 1989; J o b l i n g ,  , other " v i s c o u s "  slow down evacuation  1981; H i l t o n et  al.,  f i b e r s such as guar gum and p e c t i n  (Shiau  et  al.,  H i l t o n , Atkinson and S l i n g e r  1989).  (1983) demonstrated  s i g n i f i c a n t growth d e p r e s s i o n i n j u v e n i l e rainbow t r o u t f e d diets containing exhibited the  10 or 20% a - c e l l u l o s e .  some a d a p t a t i o n t o the f i b e r , by hypertrophy of  stomach and increased  feed  intake,  to compensate f o r the i n c r e a s e d  bulk.  (1984), on the other hand, found that can  Although the t r o u t  i t was not s u f f i c i e n t Bromley and Adkins j u v e n i l e rainbow t r o u t  compensate f o r up t o 30% d i e t a r y c e l l u l o s e by i n c r e a s i n g  their  feed  intake.  Growth and n u t r i e n t and p r o t e i n  c o n v e r s i o n e f f i c i e n c i e s were not a f f e c t e d a d v e r s e l y . t r o u t stomachs became heavier only cellulose levels. increased  bulk of the stomach i s an a d a p t a t i o n t o f i b e r ,  to handle f i b e r .  f i s h a r e more l i m i t e d i n t h e i r a b i l i t y  Chinook salmon f e d 50% c e l l u l o s e d i e t s  reduced feed  intake  (Buhler and Halver, 1961). their  feed  at 40% t o 50% d i e t a r y  These i n v e s t i g a t o r s suggested that i f  then perhaps smaller  exhibited  The  intake  and lowered p r o t e i n e f f i c i e n c y  Channel c a t f i s h a l s o  reduced  a t t h i s l e v e l of c e l l u l o s e , however, t h e i r  16  p r o t e i n conversion e f f i c i e n c y Sneed,  was not decreased.  1966).  An a d d i t i o n a l c o n s i d e r a t i o n it  (Dupree and  i n c r e a s e s the  fecal  volume,  q u a l i t y necessitating greater  with d i e t a r y  fiber  is  that  and hence lowers water a e r a t i o n and/or water  and adds to environmental p o l l u t i o n  flow,  (Bromley and A d k i n s ,  1984). 2.9  Species Comparisons  (Fish)  Carbohydrate s t u d i e s have been conducted on s p e c i e s of Despite  f i s h that possess very d i f f e r e n t  their different  dietary adaptations,  h e r b i v o r o u s and d e t r i t i v o r o u s p r o t e i n requirements,  dietary  habits.  omnivorous,  f i s h a l l have high d i e t a r y  and t h e i r a b i l i t y to d i g e s t p r o t e i n  comparable to that of c a r n i v o r e s  ( T y t l e r and Calow,  Omnivorous f i s h are b e t t e r able to u t i l i z e carnivorous species.  several  1985).  s t a r c h than  T h i s was c l e a r l y demonstrated  study by F u r u i c h i and Yone (1980) in which  is  in a  semi-purified  d i e t s c o n t a i n i n g 0 to 40% d e x t r i n were fed to f i s h of  three  s p e c i e s ; the c a r p ,  former  red sea bream, and y e l l o w t a i l  two s p e c i e s are omnivores and the l a t t e r Growth was r e t a r d e d and feed e f f i c i e n c y 40%,  sea bream at 30% and y e l l o w t a i l  is a carnivore). lowered i n carp at  at 20% d i e t a r y d e x t r i n .  As d e x t r i n and p r o t e i n were w e l l absorbed in a l l regardless  of d e x t r i n l e v e l ,  presumed.  Takeuchi et  adequate  protein  (32%)  al .  (the  species  poor u t i l i z a t i o n of d e x t r i n was  (1979) reported that c a r p fed  can e f f e c t i v e l y  utilize  carbohydrate  17  at a d i e t a r y l e v e l  of 42% without adverse e f f e c t s on growth,  feed c o n v e r s i o n or net p r o t e i n u t i l i z a t i o n efficiencies  (NPU).  of young c a r p fed a 45% a - s t a r c h d i e t  sea bream fed a 30% a - s t a r c h d i e t 74% and 47%, r e s p e c t i v e l y  ( F u r u i c h i and Yone,  1982b). the growth of  e e l s was enhanced by i n c r e a s i n g d i e t a r y glucose 30%, with a p r o t e i n l e v e l  f i x e d at 45% of the  Increased carbohydrate a l s o i n d i c a t i n g that  from 10% to  diet.  l e d to i n c r e a s e d body  fat,  i t was being used for energy and converted  I t appears that  the e e l has a higher  requirement and i s b e t t e r carbohydrate sources and V i o l a  and red  have been r e p o r t e d to be  In a study by Degani and Levanon (1987),  to f a t .  Feed  able to u t i l i z e  energy  different  i n comparison to the s a l m o n i d .  (1987) demonstrated higher s p e c i f i c  Degani  growth r a t e ,  energy r e t e n t i o n and feed c o n v e r s i o n in European e e l s {Angui11 a angui I I a) fed d i e t s with 40% p r o t e i n and 38% wheat meal, as compared to those fed 50% and 20%, or 30% and 56%, respectively. 2.9.1  S t r a i n Comparisons Chinook salmon possess great g e n e t i c d i v e r s i t y among  many d i f f e r e n t  strains,  v a r i a t i o n s in l i f e  which i s e x h i b i t e d i n wide  history patterns.  "Ocean-type" chinook  migrate to sea as u n d e r y e a r l i n g s , and "stream-type" o v e r w i n t e r i n g one year or more in f r e s h water. ocean-type  chinook enter e s t u a r i e s  as f r y and subsequently  shortly after  after  While most emergence  spend up to s e v e r a l weeks there and  18  then migrate to the ocean,  others  spend 3 months i n r i v e r s  and r e s i d e very b r i e f l y i n e s t u a r i e s before ocean entry ( C a r l and H e a l e y ,  1984).  Accompanying these d i f f e r e n c e s  migratory p a t t e r n , are d i f f e r e n c e s Larkin,  i n behaviour  in  (Taylor and  1986), growth and time of m a t u r i t y (Withler et  al.,  1987). Comparison of s t r a i n s or f a m i l i e s  for d i f f e r e n c e s  in  carbohydrate u t i l i z a t i o n have not p r e v i o u s l y been conducted on chinook salmon. c a r r i e d out  However, such i n v e s t i g a t i o n  i n two s t u d i e s on rainbow t r o u t .  (1977) examined growth performances of t r o u t from ten d i f f e r e n t containing different  families  has been  Edwards et  al.  f i n g e r l i n g rainbow  fed isonitrogenous  p r o p o r t i o n s of m e t a b o l i z a b l e  diets  energy  (17% - 38%) as c a r b o h y d r a t e . ^ Although there were significant diet,  growth d i f f e r e n c e s  between f a m i l i e s  there was no i n t e r a c t i o n between d i e t  i n d i c a t i n g u n l i k e l y prospects  and Austreng (1981),  families  and f i v e  carbohydrate, strains  of  In a companion study by  i n which f i v e  rainbow t r o u t  inbred groups were fed 15% - 49% of ME as  the prospects  for s e l e c t i v e breeding of  f o r carbohydrate u t i l i z a t i o n were once again not  very p r o m i s i n g . trout,  and f a m i l y ,  for g e n e t i c s e l e c t i o n  enhanced carbohydrate u t i l i z a t i o n . Refstie  fed the same  Despite  i t was p o s t u l a t e d  potential  for s e l e c t i o n  these negative f i n d i n g s  i n rainbow  that chinook salmon may r e v e a l some of t r a i t s a s s o c i a t e d  carbohydrate u t i l i z a t i o n owing to the  with enhanced  aforementioned  19  differences  in t h e i r  life  h i s t o r y and consequently  d i v e r s i t y and amounts of n a t u r a l prey 2.10  in  the  ingested.  Carbohydrate S t r u c t u r e The primary carbohydrate encountered  practical plant  by salmonids  c u l t u r e d i e t s i s complex carbohydrate  starch.  i n the  S t a r c h i s composed of two types of  c a l l e d amylose  and a m y l o p e c t i n ,  As examples,  form  glucosans  the p r o p o r t i o n s of  vary depending on s t a r c h source.  in  which  wheat,  potato  and o r d i n a r y corn s t a r c h are approximately 80% amylopectin and 20% amylose, 50% of  the  w i t h 0-1,4  while other  latter.  s t a r c h e s may c o n t a i n more than  Amylose i s a l i n e a r polymer of  glucoside  linkages,  while amylopectin  branched glucose polymer bonded by a-1,4 Glycogen i n l i v e r and muscle amylopectin, (Linder, 2.11  Tietz,  Digestible while  hydrolyzes  a-1,4,  During d i g e s t i o n ,  but not a - 1 , 6 ,  and  a-1,6  j3~1,4 bonds  the enzyme  glucoside  bonds.  (ie.  a-amylase This  The  latter  s i z e p i e c e s of glucose polymer  from p a r t i a l h y d r o l y s i s .  subsequently  branches.  a-1,4  formation of maltose and d e x t r i n s .  are mixtures of d i f f e r e n t resulting  to  and A b s o r p t i o n  f i b e r has a predominance of  pectins).  linkages.  i s very s i m i l a r i n s t r u c t u r e  carbohydrates c o n t a i n mostly  cellulose,  l e a d s to the  and a-1,6  1980)  Carbohydrate D i g e s t i o n  linkages,  is a  but with more e x t e n s i v e and s h o r t e r  1985;  glucose  h y d r o l y z e d , however,  Some of  these are  residual dextrins  remain  20  after  amylopectin d i g e s t i o n .  a l s o the  structure  hydrolysis  (ie.  Not only the c o m p o s i t i o n ,  branching) of a s t a r c h a f f e c t s  of a-amylase  activities  its  rate.  S t u d i e s of d i g e s t i v e enzymes i n salmonids r e v e a l presence  but  and d i s a c c h a r i d a s e s ,  but  the  their  are low in comparison to those of h e r b i v o r o u s and  omnivorous s p e c i e s (Nagayama and S a i t o , Surprisingly,  1969).  a c t i v i t y of sucrase has been shown to exceed  that of maltase  in the t r o u t  (Buddington and H i l t o n ,  1988),  and t h i s may a l s o be t r u e i n chinook salmon (Buhler and Halver,  1961).  The r e l a t i v e  efficiencies  of a b s o r p t i o n of  carbohydrate are highest for monosaccharides,  followed by  d i s a c c h a r i d e s then cooked s t a r c h , and poorest  i n raw s t a r c h ,  in brook t r o u t and Nose, 2.12  1967;  (Phillips, Smith,  1948), and rainbow t r o u t  (Singh  1971).  Carbohydrate Metabolism Salmonids e x h i b i t  prolonged hyperglycaemia when  a d m i n i s t e r e d a glucose l o a d , with plasma glucose remaining elevated 1972;  24 hours a f t e r  P h i l l i p s et  al . ,  the c h a l l e n g e 1 948;  (Palmer and Ryman,  Bergot,  1979b).  This  indicates  that they have a poor a b i l i t y to u t i l i z e c i r c u l a t i n g glucose, mammals.  a response  s i m i l a r to that observed i n d i a b e t i c  It has been shown that  u t i l i z a t i o n of glucose i n f i s h compensating  is  the  "aerobic o x i d a t i o n and  low,  even when  for temperature d i f f e r e n c e s "  Cowey and Sargent,  1979).  ( L i n et  al . ,  1978;  In s t u d i e s using r a d i o - l a b e l l e d  21  glucose,  L i n et  Bever et  al.  al.  (1978) working with coho salmon, and  (1977) with k e l p bass (Paralabrax  that the turnover r a t e s of glucose that  i n mammals  Cowey et  al.  (Katz et  al . ,  sp.)  were approximately  1976; Armstrong,  (1977a) c a l c u l a t e d the glucose  to be 13.7% of body weight,  while  value was 30% (Friedmann et  al.,  (1986) demonstrated that heat oxygen intake of  individual  found  1979).  space  in trout  i n the r a t , the determined 1965).  Beamish et  al.  increment, as measured by the  fish  i n a swimming r e s p i r o m e t e r ,  was higher i n rainbow t r o u t fed a 30% c e r e l o s e d i e t , those fed an i s o n i t r o g e n o u s consistent  with the  the r e s u l t a n t absorption, 2.12.1  1/10  18% l i p i d d i e t .  inefficient  This  than i n  is  u t i l i z a t i o n of g l u c o s e ,  i n c r e a s e d energy expenditure for  and  its  metabolism and e x c r e t i o n .  G l y c o l y s i s and Gluconeogenesis  G l y c o l y s i s i s the main pathway of carbohydrate degradation for energy and b i o s y n t h e t i c organisms  ( F i d e u , 1983).  reactions  Most of the enzymatic r e a c t i o n s of  q l y c o l y s i s are r e v e r s i b l e with the exception enzymes:  hexokinase  pyruvate kinase  pathway i s  (HK);  three (PFK); and  When f i s h are producing  through gluconeogenesis,  reversed.  of  phosphofructokinase  (PK) ( F i g u r e 1).  t h e i r own glucose  in most  the  glycolysis  The steps that are i r r e v e r s i b l e  (above) are bypassed by the corresponding enzymes, respectively:  glucose-6-phosphatase;  diphosphatase;  and pyruvate c a r b o x y l a s e  fructose (PC) i n c o n j u n c t i o n  22  F i g u r e 1.  Glucose Metabolism: g l y c o l y s i s and gluconeogenesis pathways.  Glycogen Glucose  UDP Glucose ose-6s phat as e  Hexoki nas  Glucose-6-phosphate •  —  T  Glucose-1-phosphate  Gl ucos e-6-phos  dehydrogenase  phat e  (G6PDH)  PENTOSE PHOSPHATE PATHWAY  Fructose-6-phosphate A.  Fruct ose di phos phat as e  Phosphof r uctokinase (PFK)  (FD)  Fructose-1-6-diphosphate  I I I  Phosphoenolpyruvate  Phosphoenolpyruvate  j;ar boxyki nas e (PEPCK) Oxaloacetate  Pyruvat  e Ki nas e  (PK) Lactate <  Pyruvate  > Pyruvate A c e t y l CoA  carboxylase  (PC)  23  with phosphoenolpyruvate-carboxykinase (Walton and Cowey,  (Figure  occurs p r i m a r i l y  in s k e l e t a l  and heart  gluconeogenesis i s c a r r i e d out mainly by the  (Knox et  al.,  1).  1982)  While g l y c o l y s i s muscle,  (PEPCK)  1980).  Salmonids f u l f i l l  liver.  t h e i r glucose needs  p r i m a r i l y through gluconeogenesis, as t h e i r n a t u r a l d i e t contains  little  carbohydrate.  (1981) r e p o r t e d that compared with other  Nonetheless,  "gluconeogenic  French et  activity  in trout  is  low  f i s h e s and mammals".  Before glucose can p a r t i c i p a t e i n metabolism, be phosphorylated to glucose-6-phosphate. responsible  al.  i s hexokinase,  it  must  The enzyme  a r e g u l a t o r y enzyme.  HK a c t i v i t y  has been shown to be much higher i n muscle t i s s u e than l i v e r tissue  (Shibata,  1977).  In the l i v e r s of many animals  i s an i n d u c i b l e hexokinase for g l u c o s e ,  isoenzyme,  glucokinase,  which has a high Km and i s not  product g l u c o s e - 6 - P .  With t h i s enzyme,  no isoenzyme  been d e t e c t e d al.,  glucose loads can be  1977b), and hexokinase  c a r n i v o r o u s cat to the r a t ,  fish,  enzymes.  1980;  has  Cowey et  l e v e l s are low i n comparison to Glucokinase i s a l s o absent i n the  (MacDonald and Rogers,  1984).  When compared  f i s h have a lower c a p a c i t y for p h o s p h o r y l a t i o n  of glucose i n both muscle and l i v e r .  For example,  a c t i v i t y of HK ( i n c l u d i n g glucokinase) that  In  with the p r o p e r t i e s of glucokinase  (Nagayama and Ohshima, 1974,  other g l y c o l y t i c  specific  i n h i b i t e d by the  q u i c k l y handled, and blood glucose r e g u l a t e d . however,  there  in the r a t l i v e r , and a c t i v i t y  the  in f i s h l i v e r  i n the  is  f i s h kidney  1/10 is  24  1/3  that  i n the r a t kidney (Cowey and Sargent,  and Sargent  (1979) conclude that t h i s  reasons  f i s h cannot metabolize  glucose  i s converted to g l u c o s e - 6 - P  glycolysis  Cowey  i s one of the primary  glucose  rapidly.  Once  i t can then enter  (Embden-Meyerhof pathway),  the pentose-phosphate  1979).  glycogen  into  synthesis,  pathway, or be reconverted to  glucose.  The two enzymes w i t h i n g l y c o l y s i s ' w h i c h are r e g u l a t o r y in most animals are phosphofructokinase kinase  (PK) ( F i g u r e 1).  responsible fructose  (PFK) and pyruvate  The former i s a complex enzyme  for the c o n v e r s i o n of  1,6-diphosphate.  fructose  The l a t t e r  phosphoenolpyruvate to p y r u v a t e .  6-phosphate  to  converts  Pyruvate kinase was  r e p o r t e d by Guderly and Cardenas (1980) not to be r e g u l a t o r y in the rainbow t r o u t .  PK has been found to be  higher than PFK in rainbow t r o u t  (Shibata,  10-fold  1977).  The pentose phosphate pathway i s another or secondary pathway of glucose pathway i s  The f i r s t  the dehydrogenation of glucose  enzyme glucose to the  catabolism.  6-phosphate  dehydrogenase  formation of NADPH and pentoses.  step i n  this  6-phosphate (G6PDH).  It  by the leads  NADPH i s an  important reducing agent i n b i o s y n t h e s i s  of compounds such  as f a t t y a c i d s ,  ribose  and pentoses,  especially  used i n n u c l e i c a c i d b i o s y n t h e s i s However, the clear.  (Leninger,  5-phosphate,  1982).  importance of t h i s pathway i n f i s h  is  still  not  On the one hand i t has been r e p o r t e d to be of minor  importance i n glucose while on the o t h e r ,  it  breakdown (Hochachka, 1961,  1969),  has been suggested to be the major  25  route for glucose c a t a b o l i s m in f i s h These l a t t e r  investigators  PFK a c t i v i t i e s ,  (Fideu et  al.,  1983).  concluded that due to very low  and i n d u c t i o n of  increased G6PDH a c t i v i t y  t r o u t on high carbohydrate d i e t s ,  the pentose phosphate  in  path  must be the main pathway of carbohydrate d e g r a d a t i o n .  High  l e v e l s of G6PDH have been found i n rainbow t r o u t l i v e r s , some i n v e s t i g a t o r s  have shown enhanced a c t i v i t y  high carbohydrate d i e t s Nagayama et 2.12.1.1  al.,  1973,  ( H i l t o n and A t k i n s o n ,  absorptive  gluconeogenesis i s  the c a r n i v o r e has l i t t l e  For example, relatively  less  i t s maximum i n the  state.  i t peaks hours  after  T h i s i s due to the  fact  a b i l i t y to s t o r e c a r b o h y d r a t e .  the enzymes for amino a c i d c a t a b o l i s m are  non-adaptive  in the c a t ,  to changes in d i e t a r y p r o t e i n  level  thus high n i t r o g e n l o s s i s o b l i g a t o r y even i f  dietary protein is  low (MacDonald and Rogers,  I n v e s t i g a t i o n s show that 1981).  Walton (1986) demonstrated that  versus a low p r o t e i n / h i g h carbohydrate significantly  gluconeogenic  1984).  f i s h are s i m i l a r in t h i s regard  fed a high p r o t e i n / l o w carbohydrate  exhibited  1982;  "more or  s t a t e , whereas in omnivores,  a meal in the p o s t a b s o r p t i v e  trout  fed  1975).  permanently switched on" and i s at  (Rumsey,  in f i s h  Enzyme Adaptation  In c a r n i v o r e s ,  that  and  c a t a b o l i z i n g enzymes i n v e s t i g a t e d  (60%/l0%) d i e t  (20%/56%) d i e t  lower g l y c o l y t i c  enzyme a c t i v i t i e s ,  rainbow  however,  and higher most amino a c i d  were unaffected  by d i e t .  26  H i l t o n and Atkinson alterations  i n the a c t i v i t i e s  gluconeogenic  of  key g l y c o l y t i c and  enzymes o c c u r r e d at carbohydrate l e v e l s up t o ,  but not above, diets.  (1982) found that a d a p t a t i o n v i a  14% i n rainbow t r o u t fed  These i n v e s t i g a t o r s  also  isonitrogenous  found that d i e t a r y  carbohydrate d i d not s i g n i f i c a n t l y a f f e c t c o n v e r s i o n of 1982).  [ ^ C ] a l a n i n e to glucose 1  the  percentage  ( H i l t o n and A t k i n s o n ,  T h i s poor a d a p t a t i o n of enzymes leads  obligatory nitrogen l o s s , essential  and t h u s ,  to a high  an i n a b i l i t y to  amino a c i d s when fed low p r o t e i n d i e t s .  conserve Omnivores  and h e r b i v o r e s , on the other hand, are h i g h l y a d a p t i v e . 2.12.2  Glycogen S y n t h e s i s and Breakdown  L i v e r glycogen  i n f i s h i s very s i m i l a r in s t r u c t u r e to  that found i n mammals.  In the s y n t h e s i s of glycogen ( F i g u r e  2),  i s converted to glucose  glucose  6-phosphate  UDP-glucose and e v e n t u a l l y the glucose onto the growing glycogen c h a i n . out by glycogen synthase, active  'a'  Activation  1-P,  then  u n i t s are attached  This f i n a l  step i s  carried  an enzyme which e x i s t s i n an  form (unphosphorylated) and i n a c t i v e i s brought about by a phosphatase,  i n a c t i v a t i o n by a p r o t e i n k i n a s e , as i s  'b'  form.  and  found i n mammals.  In rainbow t r o u t , the a c t i v i t y of glycogen synthase  i s some  1 5 - f o l d lower i n white muscle than i n the l i v e r and red muscle  (Ingram,  1970).  Glycogen breakdown occurs v i a a d i f f e r e n t s y n t h e s i s and i s  to  route than  r e g u l a t e d by the enzyme glycogen  27  F i g u r e 2.  L i v e r Glycogen S y n t h e s i s and Breakdown,  Glycogen A  Glyeogen Phosphor yl ase  Glyeogen Synthas e  UDP Glucose UDP glucos e phos phor ylas e  Glucose-1-phosphate Phosphogl ucomutas e Glucose-6-phosphate Hexokinas e  Glucose-6-phosphatase  Glucose  Enzymes e x i s t  i n a c t i v e and i n a c t i v e forms.  Example of r e g u l a t i o n : Glycogen  sythetase  kinase  activation  (ATP-->ADP) Hormonal > cAMP Glycogen synthetase r e g u l a t i o n by I (active > inactive) glucagon I adrenalin 4Gl ycogen phoshphoryl ase kinase a c t i v a t i o n (ATP-->ADP) Glycogen (inactive  phosphorylase > active)  28  phosphorylase,  which a l s o e x i s t s i n a c t i v e  ' a ' form  (phosphorylated in t h i s c a s e ) , and l e s s a c t i v e  'b'  form.  R e g u l a t i o n of these forms i s a l s o brought about by kinase (activation)  and phosphatase  (deactivation)  Phosphorylase breaks the a 1,4 units  i n glycogen  subsequently  glycosidic  to r e l e a s e glucose  converted to glucose  1-P,  1-P.  enzymes. link  of  which  glucose  is  Glycogen  phosphorylase enzymes are found i n l i v e r and muscle but differ  i n t h e i r s t r u c t u r e and r e g u l a t i o n .  where the purpose of glycogen  breakdown i s  In the  phosphatase  is  a c t i o n of t h i s  enzyme i s  irreversible.  fish species,  l e v e l s i n f i s h muscle  (Nagayama et  1967)  but e x i s t s al.,  i s evidence  that  in rainbow t r o u t  suggesting a d i f f e r e n t  in the l a t t e r  species.  of c a r p and g o l d f i s h ,  1972,  i n very low Shimeno and  these glycogen breakdown  ( V e r n i e r and S i r e ,  (Murat,  Amylase has been found i n the but i s only h a l f as e f f e c t i v e  i n the breakdown of l i v e r glycogen  (Murat,  Picukans and Umminger, 1979).  of  1978),  pathway of glycogen breakdown  phosphorylase 1976;  livers  as  in  goldfish  The importance  t h i s enzyme in carbohydrate metabolism in f i s h  controversial  The  I t has been found i n  phosphorylase appears to be absent i n carp l i v e r 1976),  6-  .  While there enzymes e x i s t  glucose  free  r e q u i r e d to remove the phosphate group.  the l i v e r s of s e v e r a l  Ikeda,  liver,  to l i b e r a t e  glucose i n t o the b l o o d , an a d d i t i o n a l enzyme,  they  ( C h r i s t i e n s e n and K l u n g s o y r , 1987).  is  29  White muscle, which i s  the major t i s s u e i n f i s h e s ,  p o o r l y v a s c u l a r i z e d and thus r e l a t i v e l y a n a e r o b i c . breakdown to l a c t a t e the muscle. glycolysis)  is c r u c i a l  Gluconeogenesis  L i v e r glycogen accessible  glucose  Glycogen  to maintain c o n t r a c t i o n of  from l a c t a t e  (anaerobic  and amino a c i d s i s the main source of  for muscle glycogen  is  (Moon, Walsh and Mommsen,  glucose  1985).  i s g e n e r a l l y regarded as a r e a d i l y reserve.  In r a t s i t  falls  to very low  l e v e l s w i t h i n 24 hours and i s d e p l e t e d i n 1 to 2 days (Freedland,  1967).  Fish,  r e m o b i l i z e l i v e r glycogen there  by comparison, do not (Cowey and Sargent,  is considerable inter-species  rapidly  1979), and  variation.  The c a r p and  Japanese e e l have been shown to maintain p r e - f a s t i n g c o n c e n t r a t i o n s of l i v e r glycogen a f t e r (Larsson and Lewander, 1973).  20 days without  In f a c t ,  the c a r p can be  s t a r v e d more than 100 days without d e p l e t i n g i t s glycogen  (Nagia and Ikeda,  1971).  glycogen d e c l i n e d by 80% a f t e r then i n c r e a s e d to a constant  food  liver  In rainbow t r o u t ,  20 days of  liver  feed withdrawal,  l e v e l with continued f a s t i n g ,  d u r i n g a 60 day experimental p e r i o d .  However, when p h y s i c a l  s t r e s s was imposed, l i v e r glycogen dropped at a much higher rate:  a 40% decrease  o c c u r r e d i n 30 minutes then new  synthesis quickly replenished i t al . ,  1982).  i n 45 minutes  (Morata et  30  2.12.2.1  L i v e r Glycogen  Studies  Feeding h i g h carbohydrate d i e t s to f i s h can lead substantial glycogen  elevation  in hepatic  glycogen  sugars and s t a r c h e s 1982;  Halver,  Bergot,  1961;  1979a; Palmer and Ryman,  P h i l l i p s et  al.,  1948).  goldfish  1972;  elevated fat  digestible  Several reports  indicate  and impairment of  (Dixon and H i l t o n , P h i l l i p s et  hepatomegaly,  1981;  that  dietary  al.  liver  function  in t r o u t  (1948) r e p o r t e d abdominal  Hilton, swelling,  and i n c r e a s e d m o r t a l i t y in brook t r o u t  increase  carbohydrate.  in l i v e r glycogen  1979;  sublethal  livers.  H i l t o n and A t k i n s o n , 1982;  12 % d i e t a r y d i g e s t i b l e  Bergot,  glycogen,  l e v e l s are  accompanied  ( R e f s t i e and A u s t r e n g ,  Buhler and H a l v e r , 1961).  However,  Hilton  1981,  term  1985).  (1982) demonstrated  water temperature both a f f e c t weight d u r i n g s t a r v a t i o n  1981;  possible  e f f e c t s c o u l d be d e t r i m e n t a l over a longer  (Dixon and H i l t o n ,  fed  In other  i n v e s t i g a t i o n s higher m o r t a l i t y r a t e s have not the  dextrin  carbohydrate l e v e l s above 20% leads to excess  l i v e r glycogen  only  Buhler and  in t h e i r  The e f f e c t s of e l e v a t e d l i v e r glycogen l a r g e l y unknown.  H i l t o n et  fed 25% white maize  of p r o t e i n and d e p o s i t s of  other  Palmer and Ryman  for 51 days e x h i b i t e d hyperglycaemia, depletion  fed  d e x t r i n and ( l e s s so)  ( H i l t o n and A t k i n s o n , 1982;  (1972) a l s o found that  1982).  Liver  accumulation has been r e p o r t e d in salmonids  d i e t s supplemented with g l u c o s e ,  al . ,  levels.  to  that p r e - s t a r v a t i o n d i e t  l i v e r glycogen  in rainbow t r o u t .  level In t h i s  and  and l i v e r  31  investigation,  l i v e r glycogen and l i v e r weight  with i n c r e a s i n g d i e t a r y c e r e l o s e l e v e l d e c r e a s i n g temperature. reached over  L i v e r glycogen  (0 to 34%) and levels,  which had  10 %, and l i v e r weight both d e c l i n e d to c o n t r o l  l e v e l s between 7 and 12 days a f t e r 2.13  increased  feed w i t h d r a w a l .  Endocrine C o n t r o l  2.13.1  Insulin  Insulin  i s a hormone i n v o l v e d in anabolism,  energy storage from ingested functions  include:  lipogenesis, deposition  increased  tissue.  I n s u l i n t a r g e t s the  In f i s h ,  its  liver,  primary s t r u c t u r e  (Cowey et  1977).  studies,  t i s s u e glycogen has a c t u a l l y decreased injection  in f i s h  al.,  deposition  (Ince,  i s not c l e a r ,  is  very  1983).  (Ince,  inhibit  Its  e f f e c t on  and i n some after  It appears to i n c r e a s e  c l e a r a n c e of glucose v i a o x i d a t i o n rather than  Fish differ  muscle  in mammals ( C h r i s t i e n s e n and K l u n g s o y r ,  glycogen  the  glycogen  1983). from mammals i n t h e i r a b i l i t y to  carbohydrates p a r t i c u l a r l y with respect control.  glycolysis,  As in mammals, i n s u l i n has been shown to  deposition  storage  and increased uptake of aqjino a c i d s and t h e i r  gluconeogenesis in f i s h  insulin  anabolic  of glucose i n t o  a c i d uptake i n a d i p o s e ,  s i m i l a r to that 1987).  or s h i f t  into proteins.  and adipose  Its  p e r i p h e r a l glucose u t i l i z a t i o n ,  of glucose i n glycogen enhanced f a t t y  food energy.  directing  Many i n v e s t i g a t o r s  to  metabolize  endocrine  have a s c r i b e d the poor  32  u t i l i z a t i o n of d i e t a r y glucose by f i s h to an of  i n s u l i n and c o n s i d e r  f i s h to resemble Type-I  due to i n s u l i n d e f i c i e n c y al . ,  1977a,  insufficiency  (Palmer and Ryman,  1972;  1977b; F u r u i c h i and Yone, 1981).  been shown to exert  diabetics  I n s u l i n has  some c o n t r o l over blood glucose i n  F a s t i n g hyperglycaemia has been observed i n f i s h removal of the pancreas or i s l e t s , i s l e t s with c y t o t o x i n s  (Matty,  exogenous mammalian or p i s c i n e hypoglycaemic  Cowey et  response  pronounced when f i s h  or treatment  1985).  fish.  following of  Injection  the  of  i n s u l i n causes a  in f i s h ,  and the e f f e c t  i n s u l i n i s used  (Ince,  i s more  1983).  In a  1972 study by Palmer and Ryman, i n t r a c a r d i a l a d m i n i s t r a t i o n of  insulin B . P . resulted  trout,  i n marked hypoglycaemia i n rainbow  and a d m i n i s t r a t i o n of  i n s u l i n simultaneous  glucose l o a d i n g improved glucose t o l e r a n c e Ryman,  1972).  (Palmer and  Work by these authors a l s o suggests that  mechanism of a c t i o n of  insulin stimulation  f i s h than i n mammals (Palmer and Ryman, S t i m u l a t i o n of different  with  i n s u l i n in f i s h  metabolites  is different  the in  1972).  i s brought about by  than i n mammals.  Thorpe (1976)  showed  c o r r e l a t i o n s between amino a c i d and i n s u l i n l e v e l s i n cod and rainbow t r o u t , secretion.  but found no e f f e c t  In 1977,  of glucose on i n s u l i n  Ince and Thorpe compared e f f e c t s of  v a r i o u s amino a c i d s on in perfused European e e l  vitro  in  pancreas and found that a l l amino  a c i d s t e s t e d were more e f f e c t i v e s t u d i e s using t o a d f i s h  i n s u l i n secretion  islet  than g l u c o s e .  In  t i s s u e have shown that  vitro high  33  l e v e l s of glucose Cahill,  1968).  stimulate  i n s u l i n release  However, the f a c t  that  (Tashima and  glucose  a d m i n i s t r a t i o n leads to hyperglycaemia i n d i c a t e s a l i m i t e d in  vivo  c a p a c i t y for i t s  (1982) demonstrated that carbohydrate-rich diet  release.  H i l t o n and Atkinson  feeding rainbow t r o u t a  for s e v e r a l months d i d not a f f e c t  the  s i z e or number of i n s u l i n s e c r e t i n g /3-cells i n the pancreas. In t o a d f i s h ,  glucose  was found to act s y n e r g i s t i c a l l y on  l e u c i n e s t i m u l a t i o n of 1971).  i n s u l i n secretion  (Patent and F o a ,  Huth and Rapoport (1982), however,  stimulatory effect  by l e u c i n e or glucose  production in carp.  found no  on i n s u l i n  Ince and Thorpe (1977) demonstrated  that glucose doses exceeding  100 mg/kg body weight do not  further  s t i m u l a t e the s e c r e t i o n of  (Angui11  a angui 11 a).  i n s u l i n i n the s i l v e r  eel  It has been suggested that glucose may  f u n c t i o n only to maintain b a s e l i n e  i n s u l i n l e v e l s in f i s h ,  while amino a c i d s are the main r e g u l a t o r s of p r o d u c t i o n and s e c r e t i o n In c o n t r a s t , H i l t o n  insulin  ( C h r i s t i a n s e n and Klungsoyr, et  al . (1987b)  1987).  more r e c e n t l y found  that plasma i n s u l i n was s i g n i f i c a n t l y higher in rainbow t r o u t fed a h i g h - c a r b o h y d r a t e (25% glucose) carbohydrate d i e t , insulin secretion  i n d i c a t i n g that glucose in t r o u t .  versus a lowstimulates  These authors suggested that  the t r o u t are not i n s u l i n - d e f i c i e n t and they may be more s i m i l a r to t y p e - I I et  al.,  1987b).  ( n o n - i n s u l i n dependent)  diabetics  (Hilton  34  V a r i o u s techniques  have been developed  i n s u l i n l e v e l s in mammals and f i s h . conducted using radioimmunoassays antibodies  against  subsequently i n s u l i n in  to measure  Many s t u d i e s have  (RIAs).  In t h i s  r a d i o l a b e l l e d , are used to d e t e c t plasma Initially,  i n the RIAs developed  measuring c i r c u l a t i n g hormone l e v e l s i n f i s h , mammalian i n s u l i n was used. quantitative  results  Subsequently,  (Plisetskaya  et  al.,  i t has been found that  the  to  accurate  1976). i n s u l i n of  salmon  weakly with a n t i s e r a to mammalian i n s u l i n  crossreaction 1985).  distantly  for  antisera  T h i s d i d not g i v e  commonly used i n RIAs, and bovine  al.,  method  i n s u l i n , produced in a host animal and  vitro.  crossreacts  been  i n s u l i n has no  with a n t i s e r a to f i s h The use of  fish  insulin (Plisetskaya  et  i n s u l i n of the same or even  r e l a t e d s p e c i e s has improved the assays  considerably.  R e s u l t s may be s e v e r a l  homologous RIA i s used  (Plisetskaya  f o l d higher when a  et  al.,  1986).  R e s t i n g plasma i n s u l i n l e v e l s i n salmon have been found to be i n the area of those of mammals.  4-6 n g / m l , which i s much higher than  High c i r c u l a t i n g plasma i n s u l i n  have a l s o been found i n other (1979) suggested that in f i s h  et  al.,  Temperature i s  for i t s  lower  al.  level  potency.  1986). l i k e l y to a f f e c t  for example,  very slow at low  Muggeo et  the higher c i r c u l a t i n g i n s u l i n  i s probably a compensation  (Plisetskaya  insulin,  fish species.  levels  the  f u n c t i o n of  p r o i n s u l i n conversion to  temperatures.  insulin  is  35  2.13.2  Glucagon  In mammals, glucagon  functions  p r i m a r i l y v i a breakdown of  effect  blood sugar  and a l s o by  In f i s h the hormone e x e r t s  p r i m a r i l y through s t i m u l a t i o n of  (Hayashi and O o s h i r o , 1985; 2.13.3  increase  l i v e r glycogen,  s t i m u l a t i o n of g l u c o n e o g e n e s i s . its  to  gluconeogenesis  T y t l e r and Calow,  1985).  T h y r o i d Hormone  T h y r o i d hormone has a c a l o r i g e n i c  effect  demonstrated by oxygen consumption s t u d i e s . effects  on oxygen consumption are unclear  1985).  Hilton  et  al.(1987b)  3,5,3'-triiodo-L-thyronine  found that  i n mammals, In f i s h ,  supplementation  of  the  utilization  of glucose i n rainbow t r o u t and had no  significant  effect  2.14  its  ( T y t l e r and Calow,  (T3) d i d not enhance  on plasma i n s u l i n  as  levels.  O r a l Glucose T o l e r a n c e Glucose t o l e r a n c e  i s determined by the  "rate at which  inherent mechanisms  for removing excess glucose from the  blood perform t h e i r  functions"  tolerance  test,  the d e t e c t i o n metabolism.  1984).  The glucose  or GTT, i s a common d i a g n o s t i c  t e s t used  of d i a b e t e s or other d i s o r d e r s of  glucose  In t h i s  monitored subsequent glucose.  (Linder,  test,  the s u b j e c t ' s blood glucose  is  to a d m i n i s t r a t i o n of a l a r g e dose of  In t e s t i n g humans, the usual procedure i s  to  a d m i n i s t e r a 100 g dose of glucose o r a l l y f o l l o w i n g an  for  36  overnight  fast,  then measure blood glucose at  specific  i n t e r v a l s afterward. The shape of the r e s u l t i n g blood glucose curve height  and time of occurrence of blood glucose peak)  determined by s e v e r a l of  sufficient  presence of  (ie.  factors.  i n s u l i n ; the e f f e c t i v e n e s s of  of other  factors  i n s u l i n ; the rate of  i n s u l i n antagonists; substances,  subjects,  and s e c r e t i o n  of  (Linder,  elevated  fall  1984).  i n blood In human  Return to the  105 mg/dl normally takes 1 to 2 hours  Glucose i n t o l e r a n c e and d i a b e t e s are i n d i c a t e d by r e s t i n g blood glucose l e v e l s ,  if  a higher than normal  blood glucose exceeds 180 m g / d l , the  resorption capacity urine  (Linder,  is  surpassed,  In  kidney's  and glucose i s  lost  in the  1984).  Other t e s t s such as the conducted to evaluate insulin,  30  resting  and/or delayed peak and a delayed r e t u r n to normal. humans,  of  higher than 160 mg/dl at  to 60 minutes post GTT i s abnormal.  to o c c u r .  the  counterregulatory  which stop the  i n s u l i n has acted  of 70 -  insulin;  i n s u l i n breakdown; the presence  a blood glucose l e v e l  glucose l e v e l  secretion  i n v o l v e d i n the a c t i o n and b i n d i n g  such as glucagon,  glucose a f t e r  These i n c l u d e : the  is  i n s u l i n tolerance  t e s t may be  i n d i v i d u a l s that are r e s i s t a n t  to  or have other endocrine d i s o r d e r s .  Many i n v e s t i g a t o r s f i s h equating  have reported glucose i n t o l e r a n c e  i t with type I d i a b e t e s in humans.  Ryman (1972) conducted o r a l glucose t o l e r a n c e y e a r l i n g rainbow t r o u t .  Persistent  in  Palmer and  t e s t s on  hyperglycaemia  resulted,  37  with blood glucose r i s i n g from i t s  fasting  level  of  approximately 80 mg/dl to over 500 mg/dl i n 7 hours, and remaining s l i g h t l y e l e v a t e d a d m i n i s t r a t i o n of significantly.  at 24 h o u r s .  i n s u l i n with glucose improved t o l e r a n c e  Interestingly,  prespawning females  a markedly improved o r a l glucose  in channel c a t f i s h  using s e v e r a l  exhibited  tolerance.  Wilson and Poe (1987) examined o r a l  maltose  Simultaneous  sugars.  produced the most hyperglycaemic  glucose'tolerance While glucose and response,  a d m i n i s t r a t i o n of d e x t r i n l e d to a more gradual r i s e plasma glucose due to i t s  slower d i g e s t i o n / a b s o r p t i o n  The authors suggested that  this  allows  expedite the u t i l i z a t i o n of the c i r c u l a t i n g Response curves for sucrose  to g l u c o s e .  These r e s u l t s  although b e t t e r salmonids,  and f r u c t o s e  fructose  and i t s  and thus  glucose.  were delayed due lack of  catfish,  carbohydrate then  s t i l l do not use monosaccharides and  d i s a c c h a r i d e s w e l l as energy  sources.  F u r u i c h i and Yone (1981) measured plasma i n s u l i n d u r i n g GTT i n c a r p , that  to  conversion  i n d i c a t e that channel  able to t o l e r a t e  rate.  the absorbed glucose  to synchronize with the peak i n s u l i n s e c r e t i o n  the poor a b s o r p t i o n of  in  red sea bream and y e l l o w t a i l ,  they peaked 2 hours a f t e r  the o r a l glucose  p a r a l l e l i n g plasma glucose l e v e l s .  levels  and found challenge,  They suggested that  the  i n s u l i n p a t t e r n was very s i m i l a r to that of a d i a b e t i c human. Yone,  Further investigation 1982a,  by these workers ( F u r u i c h i and  1982c) l e d them to conclude that  insulin  38  insufficiency  was the cause of poor  carbohydrate  utilization. F u r u i c h i and Yone (1982b) conducted tolerance  t e s t s in red sea bream using g l u c o s e , d e x t r i n or  a-starch.  At two hours a f t e r  which i n s u l i n s e c r e t i o n al.,  1963), 95% of  administration,  the g l u c o s e ,  absorbed d u r i n g the next 3 to peak a b s o r p t i o n o c c u r r i n g at  65% of  It  at  (Momose et  the d e x t r i n and only  Most of the  10 hours a f t e r 5 to 7 hours.  group, blood sugar peaked before absorption.  the p o i n t  has been shown to peak  4% of the s t a r c h had been absorbed.  starch,  carbohydrate  s t a r c h was  administration; In the  starch  the p o i n t of maximum  was suggested that the delayed a b s o r p t i o n of  occurring after  insulin secretion,  s t a r c h to be w e l l u t i l i z e d  allowed  ( F u r u i c h i and Yone,  the  1982b).  39  3  GENERAL MATERIALS AND METHODS  3.1  H i s t o r y and Maintenance of Experimental F i s h Between March 23 and 30,  of  each of  10 s t r a i n s  {Oneorhynchus  1987,  approximately 500  fish  of B . C . chinook salmon f r y  tshawytscha)  were brought to the Department of  F i s h e r i e s and Oceans West Vancouver Laboratory in West Vancouver. Capilano,  The stocks i n c l u d e d s i x  coastal  chinook  Big Qualicum, Quinsam, Robertson Creek, N i t i n a t  and H a r r i s o n ,  and four i n l a n d chinook s t r a i n s ;  Shuswap, Eagle R i v e r and C l e a r w a t e r .  Quesnel,  Upon a r r i v a l ,  weighed between 1 and 3 grams, the Quesnel stock slightly  higher  (approx.  2 to 4 grams).  in a 1100 1 oval f i b e r g l a s s lid,  approximate flow rate of were fed to s a t i a t i o n feeding•frequency  15-20  1/min.  3 times d a i l y ,  Each stock  (11°C)  initially,  was h e l d  at an  and  angui 11 arum)  pellets  then  was reduced to 2 times d a i l y in  stocks were d i p - v a c c i n a t e d a g a i n s t  the  September.  Vibriosis  (ordalii  at approximately 8 grams body weight. readiness  were sampled for weight.  1987,  fish  averaging  Oregon moist  All  To determine  the  tank covered with a nylon mesh  and s u p p l i e d with f r e s h w e l l water  degree of  strains;  for s m o l t i f i c a t i o n ,  A l s o , they were observed  s i l v e r i n g and jumping behaviour.  sea water t r a n s f e r  the  was i n i t i a t e d  On June  fish for 12,  in the Quinsam,  C a p i l a n o , H a r r i s o n , Robertson Creek, N i t i n a t and Quesnel strains.  The average  to 7.0  except for the Quesnel s t o c k ,  g,  weight of these f i s h ranged from 6.5 which averaged  9.5  40  g.  The Qualicum and Shuswap stocks were s t a r t e d on June 26,  when t h e i r average weights approached 7.0 and Clearwater on J u l y 23, of  6.5  shifted  and 5.5  g,  respectively.  from f r e s h water  p e r i o d of  12 to  Burrard I n l e t  when the  15 days.  g, and Eagle River  f i s h weighed an average  The f i s h were g r a d u a l l y  (FW) to sea water  (SW) over a  The sea water was pumped in from  at a depth of 30 m, and the J u n e - J u l y  temperature was 10 to  10.5°C.  The i n l a n d s t r a i n s , marine e n t r y ,  not normally accustomed to  early  e x h i b i t e d v a r y i n g degrees of d i f f i c u l t y  adapting to seawater. which r e s u l t e d  Clearwater chinook f a i l e d to  i n severe m o r t a l i t y and s t u n t i n g .  d i s c o v e r e d sometime  (months)  later  in  smoltify  It  was  that many of the Eagle  R i v e r and Shuswap f i s h had a l s o f a i l e d to p r o p e r l y  smoltify,  resulting  reversion  i n growth r e d u c t i o n , some s t u n t i n g ,  and i n c r e a s e d  disease.  Experiments were d e l a y e d , following  unfortunately,  s p r i n g (1988), when new l a b o r a t o r y  became a v a i l a b l e periodically  parr  for e x p e r i m e n t a t i o n .  sampled for weight,  until  the  facilities  The stocks were  c u l l e d to some e x t e n t , and  d i v i d e d among a few a d d i t i o n a l tanks where  possible.  Feeding was reduced to l i m i t growth d u r i n g p a r t of the and winter due to r e s t r i c t e d tank space.  fall  Sea water  temperature reached 13°C i n September and dropped to about 7°C in February 1988.  D i s s o l v e d oxygen  (DO) values  between 6 and 8 p p t , and were lowest a f t e r S a l i n i t y ranged from 21 to 30 p p t .  varied  feeding.  L i g h t i n g followed  the  41  natural photoperiod. with o x y t e t r a c y c l i n e sulphamerazine  The chinook stocks were t r e a t e d once h y d r o c h l o r i d e and once with  due to outbreaks of  In e a r l y February of  Vibriosis.  1988 the nine remaining stocks  were moved to 3.0 m diameter  7500 1) outdoor c i r c u l a r  tanks with a flow r a t e s of approximately 35 1/min. 16,  1988,  these stocks were moved to the new DFO f a c i l i t y  i n t o 2.5 m diameter  (<=* 6000 1) outdoor c i r c u l a r t a n k s ,  the same seawater system, experimentation. shifted (4.0  on  where they remained u n t i l  Shortly after  from the OMP d i e t  mm).  On March  (3.0  By the end of A p r i l ,  this  time a l l f i s h were  mm) to a commercial dry d i e t the s t r a i n average  v a r i e d from 53 g (Harrison) to 92 g  (Quesnel).  weights  42  4  EXPERIMENT 1. The E f f e c t s of Feeding a High Versus Low Carbohydrate D i e t on Growth, Body Composition, Feed and P r o t e i n U t i l i z a t i o n , L i v e r Glycogen C o n c e n t r a t i o n and L i v e r Weight in S e l e c t e d S t r a i n s of B. C . Chinook Salmon.  4.1  MATERIALS AND METHODS  4.1.2  D i e t P r e p a r a t i o n and Composition The compositions  of the two t e s t d i e t s ,  " c o n t r o l " and "high carbohydrate" d i e t s , 1.  r e f e r r e d to as  are given  i n Table  The t e s t d i e t s were formulated to be i s o n i t r o g e n o u s  g p r o t e i n / k g d i e t ) and i s o c a l o r i c  (3633 k c a l ME/kg)  (440  (see  Table 2 ) . D i e t s were p e l l e t e d under reduced steam pressure California Pellet  Mill  in a  through a 5/32" d i e then a i r - d r i e d .  As the h e r r i n g o i l content of the c o n t r o l d i e t would have been too h i g h for p e l l e t i n g , excluded  from the mash.  a p o r t i o n of the o i l was  After p e l l e t i n g ,  was sprayed onto the c o n t r o l d i e t d i e t s were stored frozen at  -18°C.  in p l a s t i c  the remaining o i l  i n a r o t a t i n g drum.  bags i n s i d e  The  paper bags and kept  43 Table 1 - Composition of  t e s t d i e t s as  fed.  LC D i e t  HC D i e t  1  (g/kg) Steam-dried h e r r i n g meal F r e e z e - d r i e d euphausids^ P o u l t r y - b y - p r o d u c t meal a-cellulose P r e - g e l a t i n i z e d wheat s t a r c h Herring o i l (stabilized) Vitamin supplement M i n e r a l supplement^ Permapell C h o l i n e c h l o r i d e (60%) Ascorbic a c i d Chromic oxide  (g/kg)  387.86 155.29 120.83 185.71  387.86 155.29 120.83  -  286.97  -  101.26 1 4.35 9.57 1 3.66 4.78 1.91 4.78 1000.00  4  -  14.35 9.57 13.66 4.78 1 .91 4.78 1000.00  L C : low c a r b o h y d r a t e / h i g h l i p i d ( c o n t r o l ) d i e t . HC: h i g h carbohydrate/low l i p i d d i e t . S t a b i l i z e d with 200 ppm ethoxyquin Vitamin supplement s u p p l i e d the f o l l o w i n g l e v e l s of n u t r i e n t s per kg of d i e t as f e d : D-calcium pantothenate, 185.2 mg; p y r i d o x i n e H C l , 43.0 mg; r i b o f l a v i n , 57.4 mg; n i a c i n , 286.8 mg; folic acid, 19.1 mg; thiamin mononitrate, 38.8 mg; biotin, 2.87 mg; cyanocobalamine, 57 ug; menadione 25 mg; d l - a - t o c o p h e r y l a c e t a t e , 573.6 IU; c h o l e c a l c i f e r o l , 2,294.4 IU; r e t i n o l a c e t a t e , 9,560.0 IU; i n o s i t o l , 382.4 mg. M i n e r a l supplement s u p p l i e d the f o l l o w i n g l e v e l s of n u t r i e n t s in mg/kg of d i e t as f e d : c o b a l t (as C o C l * 6 H 0 ) , 0.96; copper (as C u S 0 « 5 H 0 ) , 3.29; iron (as F e S 0 « 7 H 0 ) , 40.2; magnesium (as M g S 0 « 7 H 0 ) , 371; manganese (as M n S 0 « H 0 ) , 84.9; selenium (as N a S e 0 ) , 0.096; z i n c (as ZnS0 >7H 0), 65.7; iodine (as K I 0 ) , 4.8; f l u o r i n e (as NaF), 4 . 3 . 2  2  4  4  2  4  4  2  3  2  4  3  2  2  2  44 Table 2 - C a l c u l a t e d m e t a b o l i z a b l e d i e t s on a m o i s t u r e - f r e e  Energy Source  k c a l ME per kg  energy v a l u e s of basis.  LC D i e t 1  HC D i e t  g/kg  g/kg  k c a l ME per kg  test  1  k c a l ME per kg  Protein  4.5  2  440.0  1980  Lipid  8.5  2  187.5  1594  CHO-animal -gelat. starch  3.8  2  15.6  59  15.6  59  3.0  3  -  300.0  900  T o t a l ME  —  440.0 81 .64  3633  LC D i e t = low carbohydrate ( c o n t r o l ) d i e t ; HC D i e t = high carbohydrate d i e t . From Beamish et al . (1986). Assumed 75% d i g e s t i b i l i t y at t h i s d i e t a r y i n c l u s i o n (Singh and Nose, 1967).  1980 694  3633  level  45  4.1.3  Fish Selection Two c o a s t a l  and Tank A l l o c a t i o n  chinook s t r a i n s ,  Robertson Creek and B i g  Qualicum, and two i n l a n d chinook s t r a i n s , Shuswap, were s e l e c t e d for t h i s  study.  Quesnel and  On May 28,  f i s h were randomly s e l e c t e d as a r e p r e s e n t a t i v e each of these s t r a i n s , (0.35  ml/1)  anesthetized  in  and weighed to the nearest  differences  i n growth r a t e s ,  1988,  sample from  2-phenoxyethanol 0.1  g.  Due to  the s t a r t i n g weights and weight  d i s t r i b u t i o n s were not homogeneous between s t r a i n s . t o t a l number of experimental was as f o l l o w s ;  120 Robertson Creek,  two s t r a i n s ) .  120 B i g Qualicum,  (there were fewer  tanks.  Creek,  4 tanks of  tank had 27) 23)  3  to 4.5  30 B i g Qualicum,  4 tanks of  Quesnel, and 4 tanks of 24 (note:  kg/m . 3  ranged from 10 °C to  arranged.  26 (note: 1 1 tank had  Each tank was provided with a e r a t i o n 10 1/min.  Water temperature  13 °C over the course of  L i g h t i n g followed  the  the n a t u r a l p h o t o p e r i o d .  The 16 experimental tanks were s i t u a t e d with the  16  I n i t i a l d e n s i t i e s ranged from 4.0  and a seawater flow rate of  experiment.  of  there were 4 tanks of 30 Robertson  Shuswap chinook.  kg/m  f i s h of  800 1 indoor  T h e r e f o r e , out of a t o t a l  experimental tanks,  105  Each stock was randomly d i v i d e d  i n t o 4 equal groups p l a c e d i n separate fiberglass  The  f i s h s e l e c t e d from each s t r a i n  Quesnel, and 96 Shuswap chinook the l a t t e r  50  i n 2 rows of  four groups of chinook from each s t r a i n randomly  8,  46  Experimental f i s h were a c c l i m a t e d for 20 days, d u r i n g which time they were g r a d u a l l y s h i f t e d dry d i e t 4.1.4  to a c o n t r o l d i e t ,  from the commercial  over a 7 day p e r i o d .  S t a r t of Experiment Each experimental  individual withheld  sampling was c a r r i e d out on  rows, over two c o n s e c u t i v e  1 day p r i o r to weighing the  (Day 0 of experiment),  each group of  days.  Feed was  fish.  On June 17,  fish  from row 1 was  1988  captured and d i v i d e d among three  30 1 p l a s t i c  aerated seawater.  f i s h from each tank were  singly  The f i r s t  k i l l e d by an overdose  phenoxyethanol), measured (to livers  weighed  the nearest  six  of a n e s t h e t i c  (to the nearest 0.1  cm).  r a p i d l y removed, weighed  buckets of  (0.8  0.01  ml/1  g) and  Four f i s h had t h e i r  (± 0.0001 g ) ,  flash  in l i q u i d n i t r o g e n , and s t o r e d at - 4 0 ° C for l a t e r determination. on each c a r c a s s , kidney,  examining major organs,  f o r any evidence  proximate a n a l y s i s .  the  frozen  glycogen  A general h e a l t h check was then c a r r i e d out  of d i s e a s e .  especially  subsequent  The remaining f i s h were  2-phenoxyethanol),  the  Two f i s h were frozen  whole on dry i c e then s t o r e d at - 1 8 ° C for  (0.35  2-  anesthetized  weighed and measured.  On June,  18,  f o l l o w i n g day, the second row of experimental groups was  sampled i n the same manner. control diet  to t h i s p o i n t ,  As a l l  f i s h had been fed  the  samples  from two p a i r s of  tanks  c o n t a i n i n g the same chinook s t r a i n , were p o o l e d .  47  Experimental feeding commenced on June 18 for row 1 and June 19 for row 2.  Two of the  four experimental groups of  chinook w i t h i n each s t r a i n were continued on the c o n t r o l (LC) d i e t (HC) d i e t . 9:00  to  and the other two were fed the high carbohydrate A l l f i s h were fed twice d a i l y to s a t i a t i o n ,  10:00  am and from 3:00  to 4:00  intake was recorded for each group.  pm.  Daily  from  feed  Feed was w i t h h e l d one  day p r i o r to sampling. F i s h weights and lengths were again recorded on day 21 ( J u l y 7 and 8, 28 and 29) the  rows 1 and 2 r e s p e c t i v e l y )  of the experiment.  On day 63 (August  f i n a l day of the growth t r i a l ,  each tank were k i l l e d .  the f i r s t  E i g h t f i s h had t h e i r  and q u i c k - f r o z e n i n l i q u i d n i t r o g e n , whole on dry i c e  For the ensuing 21 days, remaining f i s h .  18 and  19),  12 f i s h from livers  removed  and the remaining  previously.  feed was w i t h h e l d from a l l  On Sept 8 and 9,  the  first  from each tank were s a c r i f i c e d for l i v e r removal, as All  (July  4 f i s h were frozen  for proximate a n a l y s i s ,  f i s h were weighed and measured, as  groups of  and day 42  8 fish before.  f i s h weights and lengths were r e c o r d e d .  4.1.5  Proximate Body Composition This analysis  consisted  of dry matter,  crude l i p i d and ash determinations from each treatment trial.  of the  crude p r o t e i n ,  fish  carcasses  group on days 0 and 63 of the  growth  The frozen whole chinook had been s t o r e d at - 1 8 ° C  for approximately 4 months.  Each sample,  consisting  of 4  48  fish,  was  removed affect  chopped  from  t h e stomach and  the r e s u l t s .  a meat g r i n d e r , m i x t u r e was then  into pieces while  The  2 or  freeze-dried  3 times.  The  f o r 72 h o u r s .  Each  All  a n a l y s e s were p e r f o r m e d  patty  drying  not  and  refrozen, sample  coffee  in triplicate.  through  paste-like  freeze-dried  t o a f i n e powder w i t h a Braun  after  was  i t would  resulting  thick  reground  so  Feed  s e c t i o n s were g r o u n d  1 cm  was  determined  intestine,  frozen  pressed into a  frozen.  Dry  t h e powdered samples  grinder.  matter  was  f o r 24 h o u r s  at  85°C. Lipid B l i g h and g of  c o n t e n t was Dyer  (1959) e x t r a c t i o n  freeze-dried  a blender with water.  ground  10 ml  Another  f o l l o w e d by  teaspoon  of  flask The  was  flask.  After  was  then  graduated  cylinder.  f o r three minutes  of b l e n d i n g .  seconds  and  glass  ml  Finally, water  were  of b l e n d i n g . glass  1/2  The  funnel into a  graduated  cylinder. 1:1  the washings added t o the  solvent  The  volume o f t h e  l a y e r s were a l l o w e d t o  the methanol 5 ml  10  t h e c o n t e n t s o f t h e vacuum  The  Triplicate  and  2.0  added t o t h e  w i t h a s m a l l amount o f  solution  r e c o r d e d and  suction.  30  filtering,  rinsed  separate overnight. was  mixed  a p p r o x i m a t e l y 5 ml  i n t o a 50 ml  chloroform:methanol  by  seconds  the  Approximately  through a s i n t e r e d  were e m p t i e d  flask  layer  30  f i l t e r - a i d and  passed  was  of  method.  of c h l o r o f o r m was  f o l l o w e d by a n o t h e r  mixture vacuum  fish  by a m o d i f i c a t i o n  c h l o r o f o r m , 20 ml m e t h a n o l  10 ml  mixture,  added,  determined  chloroform/lipid  layer  subsamples  of  was the  then  removed  in  49  chloroform/lipid determine  l a y e r were evaporated and d r i e d i n t i n s  l i p i d weight.  Total % carcass  to  l i p i d was then  calculated. P r o t e i n content was determined by the technique  using a Technicon A u t o a n a l y z e r .  a l i q u o t of each sample was d i g e s t e d for  A 0.1  After cooling,  to the m i x t u r e , b r i n g i n g the t o t a l  0.15  and 2.0 ml  d i s t i l l e d water was added volume to 50 m l .  were then run through the a u t o a n a l y z e r , and n i t r o g e n s t a n d a r d s .  to  1 hour in 10 ml of  concentrated H2SO4 with a mercury c a t a l y s t hydrogen.peroxide.  micro-Kjeldahl  Samples  along with blanks  Determination of n i t r o g e n by t h i s  procedure i s based on a c o l o r i m e t r i c method in which a green color  is  formed by the  salicylate,  r e a c t i o n of ammonia sodium  sodium n i t r o p r u s s i d e and sodium h y p o c h l o r i t e  a b u f f e r e d a l k a l i n e medium (pH 12.8 salicylate  complex  is  c o l o r i m e t e r chamber. absorbance  read at  -  13.0).  The ammonia  660 nm wavelength  P r o t e i n was c a l c u l a t e d  in  the  from sample  peaks.  Ash content was determined by p l a c i n g the ceramic c r u c i b l e s then weighing 4.1.6  in  in a muffle  the remaining  samples in  furnace at 6 0 0 ° C for 4 hours,  residue.  L i v e r Glycogen Determination L i v e r glycogen  was q u a n t i f i e d by a m o d i f i c a t i o n of  H a s s i d and Abraham (1957) anthrone procedure.  A 5.0  g piece  of  frozen l i v e r was q u i c k l y s e c t i o n e d and mixed with 3.0  of  30% (w/v)  potassium h y d r o x i d e .  After  the  20 minutes of  mis  50  digestion  i n a b o i l i n g water b a t h , 5.0 mis of ethanol  added to p r e c i p i t a t e vortexed,  c e n t r i f u g e d at  supernatants dissolve  the glycogen.  discarded.  the glycogen.  Samples were then  10000 rpm for  10 minutes,  and the  D i s t i l l e d water was added to A 2.0 ml a l i q u o t of the s o l u t i o n  slowly added to 4.0 mis of consisting  was  f r e s h anthrone  reagent,  of 0.2% anthrone i n 98% s u l p h u r i c a c i d .  mixture was vortexed and heated water b a t h , then c o o l e d .  for  In t h i s  10 minutes  step,  was  The  in a b o i l i n g  glycogen  was  h y d r o l y z e d by the a c i d and l i b e r a t e d glucose r e a c t e d with anthrone, a l l o w i n g c o l o r i m e t r i c d e t e r m i n a t i o n .  Absorbance  was read i n a Unicam SP1800 u l t r a v i o l e t Spectrophotometer a 620 nm wavelength.  Glucose content  was c a l c u l a t e d from a  standard/absorbance curve generated using a set standards, 4.1.7  ranging i n c o n c e n t r a t i o n  Statistical  of  5 glucose  from 5 to 25 u g / m l .  Analyses  Data were analyzed using the SAS a n a l y s i s procedure  at  (SAS v e r s i o n 6.03,  1988).  Each set  of  variance  of 4  experimental u n i t s w i t h i n a chinook s t r a i n was analyzed separately treatment  by one-way a n a l y s i s or c l a s s v a r i a b l e .  each s t r a i n )  was a completely  of v a r i a n c e , with d i e t as The experimental model  of  this  strain.  (for  randomized d e s i g n .  Data from Shuswap chinook were not due to s e r i o u s  the  i n c l u d e d in analyses  d i s e a s e outbreak i n a l l experimental  groups  51  4.2 4.2.1  RESULTS Proximate Analyses of D i e t s and P r o t e i n Sources The proximate compositions  carbohydrate) and d i e t p r o t e i n sources meal,  of d i e t  1 ( c o n t r o l or low  2 (high c a r b o h y d r a t e ) , and the 3  i n c l u d e d i n these d i e t s  (steam-dried h e r r i n g  f r e e z e - d r i e d euphausids and p o u l t r y - b y - p r o d u c t meal)  are given i n Tables 3 and 4, analysis  results  of d i e t s  respectively.  1 and 2 v a r i e d only s l i g h t l y  expected values  for p r o t e i n (44.0% of d i e t )  (18.75% of d i e t  1 and 8.16% of d i e t  4.2.2  The proximate from  and l i p i d  2).  Growth and C o n d i t i o n F a c t o r Figure 3 i l l u s t r a t e s  the growth of the Quesnel, B i g  Qualicum and Robertson Creek chinook over the nine week feeding p e r i o d .  Average weights are a l s o shown i n Table  5.  Data from the Shuswap s t r a i n were excluded from a l l experimental analyses (bacterial  due to a high i n c i d e n c e of BKD  kidney d i s e a s e )  apparent d u r i n g the  i n the s t o c k ,  which became  study.  Upon commencement of the growth t r i a l , s t r a i n s examined d i f f e r e d s i g n i f i c a n t l y weights,  the four chinook  in mean body  and v a r i a n c e s around the means (see  Table  5).  Therefore d i r e c t s t r a i n comparisons c o u l d not be made, and a n a l y s i s was r e s t r i c t e d to the e f f e c t s of d i e t individual  strains.  within  52  Table 3 -  Diet  Proximate analyses of d i e t s .  Treatment  1  % D.M.  % CP  1  LC  92.14  46.16  2  HC  92.10  46.34  2  % CL 19.47 9.1 1  3  % Ash 10.98 1 1 .00  LC = low c a r b o h y d r a t e / h i g h l i p i d ( c o n t r o l ) d i e t ; HC = high carbohydrate/low l i p i d d i e t . Crude p r o t e i n was estimated from n i t r o g e n (N) d e t e r m i n a t i o n , using the m a c r o - K j e l d a h l technique (CP = %N X 6 . 2 5 ) . Crude l i p i d was determined by the B l i g h and Dyer (1959) e x t r a c t i o n procedure. Note: P r o t e i n , l i p i d and ash expressed on a dry matter ba s i s. 1  2  3  53 Table  4 - Proximate a n a l y s e s of p r o t e i n sources f o r experimental d i e t s (dry matter b a s i s ) .  Feedstuff  %DM  %CP  Steam-dried H e r r i n g Meal  93.4  Freeze-dried Euphausids P o u l t r y Byproduct  3  %Ash  71.2  10.2  16.0  92.4  61 .0  14.6  13.6  95.0  65.7  17.7  12.5  1  2  %CL  Dry m a t t e r . C r u d e p r o t e i n c o n t e n t d e t e r m i n e d by t h e m i c r o - K j e l d a h l technique. C r u d e l i p i d c o n t e n t d e t e r m i n e d by t h e B l i g h and Dyer e x t r a c t i o n method ( 1 9 5 9 ) .  Figure 3 :  Growth of Chinook Salmon of Three Strains Fed Low VS High Carbohydrate Diets Over a 63 Day Period.  Time Means ± 1 standard error of the mean, N=2.  (days)  55  Table 5  -  Mean body weights of treatment groups at i n t e r v a l s d u r i n g the experiment.  Time on d i e t Strain  Diet  1  0 D  21-day  (days)  21 D  42 D  63 D  Quesnel  LC  1 34.3 ± 2.2  1 54.1 ± 0.9  178.0 ± 2.4  201.1 ± 4.4  Quesnel  HC  135.3 ± 0.9  1 54.6 ± 5.7  170.9 ± 0.3  193.7 ± 7.4  B.  Qual.  LC  108.3 ± 1.8  122.7 ±1.2  1 33.8 ± 2.3  1 46.5 ± 6.0  B.  Qual.  HC  1 06. 1 ± 4.3  112.6 ±8.1  118.0 ± 7.8  1 35.4 ± 9.9  Rob.Crk.  LC  115.7 ± 3.3  127.3 ± 3.8  143.6 ± 5.7  165.6 ± 9.2  Rob.Crk.  HC  116.1 ± 3.8  124.2 ± 2.7  1 32.8 ± 2.0  151.6 ± 6.7  Means ± SEM (standard e r r o r of the mean) D i e t LC = low carbohydrate ( c o n t r o l ) d i e t ; D i e t HC = high carbohydrate d i e t . There were no s i g n i f i c a n t e f f e c t s of d i e t on mean bodyweights of chinook salmon w i t h i n the Quesnel, B i g Qualicum or Robertson Creek s t r a i n s , at days 21, 42 or 63 of the growth t r i a l . 1  56  In the i n d i v i d u a l analyses  of v a r i a n c e , by s t r a i n ,  there were no s i g n i f i c a n t d i f f e r e n c e s on day 21, day 42 or day  63 f o r mean body weights between the high  carbohydrate  and c o n t r o l groups w i t h i n any of the three chinook tested. general  Nevertheless,  strains  chinook of each s t r a i n e x h i b i t e d a  t r e n d of reduced body weight i n groups f e d the high  carbohydrate  diet.  Average body weight gains  (Table 6)  r e v e a l e d the same t r e n d , however, d i f f e r e n c e s were not s i g n i f i c a n t due to v a r i a t i o n between r e p l i c a t e groups. S p e c i f i c growth r a t e s (SGR, [(LnW  - LnW ) * T] x 100)  2  1  over the 63 day feeding p e r i o d are shown i n Table F i g u r e 4.  7 and  S p e c i f i c growth r a t e s were c a l c u l a t e d f o r each 21  day  i n t e r v a l of the f e e d i n g t r i a l ,  for  days 0 -21, SGR-2 f o r days 21-42, and SGR-3 f o r days 42-  63) and f o r the e n t i r e experimental 63D). rates  I n d i v i d u a l analyses  between weighings (SGR-1  feeding p e r i o d (SGR-  (by s t r a i n ) of s p e c i f i c  (SGR-1, SGR-2 and SGR-3), revealed  reductions  significant  i n the second i n t e r v a l s p e c i f i c growth r a t e (SGR-  2) of high carbohydrate-fed Robertson Creek s t r a i n s significant  growth  fish,  (p<0.05).  i n both B i g Qualicum and A s i m i l a r but non-  trend was a l s o seen i n the SGR-1 of these two  s t r a i n s , and i n the SGR-2 of Quesnel chinook f e d the high carbohydrate  diet.  carbohydrate-fed  S p e c i f i c growth r a t e s (SGR-3) of high  f i s h d u r i n g the t h i r d  i n t e r v a l were  comparable to those of c o n t r o l - f e d f i s h , chinook s t r a i n s .  i n each of the  Quesnel chinook e x h i b i t e d a l e s s  57 Table 6  -  Average body weight gains (BWG), and percent BWG of treatment groups at the end of the 63-day feeding p e r i o d .  Strain  Diet  1  BWG(g) 2  %BWG  Quesnel  LC  66.6  +  2.2  49.7  Quesnel  HC  58.9  +  8.2  43.2  B.Qual.  LC .  38.2  +  4.2  35.3  B.Qual.  HC  29.5  +  5.7  27.6  Rob.Cr.  LC  50. 1 + 6.0  43. 1  Rob.Cr.  HC  35.4  30.6  +  2.8  Means ± SEM (standard e r r o r of the  mean)  D i e t LC = low carbohydrate ( c o n t r o l ) d i e t ; D i e t HC = h i g h carbohydrate d i e t . There were no s i g n i f i c a n t e f f e c t s of d i e t on average body weight gains of chinook salmon w i t h i n the Quesnel} Big Qualicum or Robertson Creek s t r a i n s .  58 Table 7  -  S p e c i f i c Growth Rates of treatment groups over 21-day i n t e r v a l s and the 63-day d u r a t i o n of the feeding t r i a l .  Strain  Diet  1  2  SGR-1  2  SGR-2  2  SGR-3  2  SGR-63D  Quesnel  LC  .725 ±.080  .759 ±.070  .636 + .031  .707 ±.007  Quesnel  HC  .692 ±.113  .540 ± . 1 32  .669 + .146  .634 ±.055  B.  Qual.  LC  .660 ±.027  .457 ±.029  .470 ±.089  .529 ±.030  B.  Qual.  HC  .31 1 ±.115  .235 ±.021  .734 ±.029  .426 ±.041  Rob.Crk.  LC  .505 ±.007  .634 ±.035  .748 ±.060  .629 ±.034  Rob.Crk.  HC  .354 ±.042  .353 ±.025  .689 ±-106  .466 ±.013  **  **  Means ± SEM (standard e r r o r of the mean) D i e t LC = low carbohydrate ( c o n t r o l ) d i e t ; D i e t HC = high carbohydrate d i e t . S p e c i f i c Growth Rate = [ (lnW2 - lnW1) + T ] x 100 where W2 = f i n a l weight (g), W1 = i n i t i a l weight (g), and T = time i n days. SGR-1 = SGR for days 0 - 2 1 ; SGR-2 = SGR for days 21 - 42; SGR-3 = SGR for days 42 - 63; and SGR-63D = SGR for days 0 - 6 3 . * * S i g n i f i c a n t at the a=0.05 l e v e l . 1  2  59  Figure 4 : Twenty-one Day Specific Growth Rates in Chinook Salmon of Three Strains Fed Low VS High Carbohydrate Diets. 1.000  LOW CARB. DIET •  o a:  0.800  *$ 2  0.600-i  o 0  u= "o S. CO  0.400 H  HIGH CARB. DIET LZ2  QUESNEL  means ± 1 s.e.m  1  0.2000.000  1  SGR-1  SGR-2  1.000  means ± 1 s.e.m.  1  BIG QUAUCUM  3 o  0.800-j  •5  0.600  SGR-3  8  o  o V: *o §. to  0.400 0.200 0.000 1.000  o  0.800-  x: %  0.600  8  0  0.400  0  'o S. CO  0.200 0.000  1  SGR-1  ROBERTSON  £1  SGR-2  SGR-3  means ± 1 s.e.m.  1  CREEK  1  11 SGR-1 Day 0-21  (A  SGR-2 Day 21-42  Means ± 1 standard error of the mean. N - 2 . •Significant at p < 0.05 1  (A  SGR-3 Day 42-63  60  pronounced d e v i a t i o n  in s p e c i f i c  groups, and no s i g n i f i c a n t Overall  specific  significantly  (SGR-63D) were not  (Table 8 ) ,  d e f i n e d as body weight  was not s i g n i f i c a n t l y  3  4.2.3  in SGR were found.  i n f l u e n c e d by d i e t .  x 100 -s- (fork l e n g t h ) , i n the  differences  growth r a t e s  Condition factor  diet  growth r a t e s between d i e t  individual strain  analyses.  Carcass composition Initial  (percent given  and f i n a l  dry matter,  i n Table 9.  (day 63) mean c a r c a s s  Carcass composition data  carbohydrate-fed f i s h r e l a t i v e the three chinook s t r a i n s  water a l s o e x h i b i t e d a s l i g h t  significant analyses:  groups.  between d i e t  i n high  Figure 5).  'trends'  i n high  were  groups i n only the  i n each  Percent body  upward tendency  These  statistically  following  f i n a l % c a r c a s s p r o t e i n i n Big Qualicum chinook  and Robertson Creek chinook fat  (% dry matter  to c o n t r o l - f e d f i s h ,  (see  are  increased body  p r o t e i n and a s h , and decreased body f a t ,  carbohydrate d i e t  compositions  crude p r o t e i n , crude fat and ash)  b a s i s ) e x h i b i t e d a c o n s i s t e n t t r e n d of  of  i n f l u e n c e d by  ( p < 0 . 0 l ) ; and f i n a l % c a r c a s s  i n Robertson Creek chinook  (p<0.05).  comparisons revealed no s i g n i f i c a n t  A l l other  differences  (p>0.05).  61 Table 8  -  Mean c o n d i t i o n f a c t o r s i n treatment groups, i n i t i a l l y and at the end of the 63-day feeding p e r i o d .  Strain - Diet  Od-Cond.F. *  63d-Cond.F.  Quesnel - LC  1 .37 ± . 0 1  1 .45 ± . 0 0  Quesnel - HC  1 .40 ± . 0 3  1 .45 ± . 0 6  B.Qual.  - LC  1 .25 ± . 0 2  1.31  B.Qual.  - HC  1.23  ±.03  1 .30 ± . 0 4  Rob.Cr.  - LC  1 .29 ± . 0 3  1 .37 ± . 0 5  Rob.Cr.  - HC  1 .30 ± . 0 1  1 .34 ± . 0 4  1  -  Means ± SEM (standard e r r o r of the  ±.03  mean)  D i e t LC = low carbohydrate ( c o n t r o l ) d i e t ; D i e t HC = high carbohydrate d i e t . C o n d i t i o n f a c t o r = body weight x 100 T (fork l e n g t h ) . There were no s i g n i f i c a n t e f f e c t s of d i e t on f i n a l (day 63) c o n d i t i o n f a c t o r s of chinook salmon w i t h i n the Quesnel, Big Qualicum or Robertson Creek s t r a i n s . 3  62 Table 9  S-D  2  -  %Dry Matter Od  Qs-LC  Proximate a n a l y s e s of whole  4  63d  \ 24.84 ±.27  27.36 ±.51  Qs-HC  /  26.55 ±2.03  BQ-LC  \ 22.67 ±.34  25.79 ±.29  /  24.72 ±.62  \ 24.24 ±.45  27.07 ±.06  BQ-HC RC-LC RC-HC  /  25.03 ±.80  %Protein Od 61 .96 ±.45  68.06 ±.61  64.45 ±1 .23  %Lipid  3  63d  Od  58.35 ±1 .91  63.00 ±.10 65.58 ±.08  15.49 , .^ ± . 9 4 K -X  58.72 ±.48 65.97 *** ±.05  18.86 ±1-  %Ash  3  63d  23.03 ±.66  60.84 ±2.59  Means ± SEM (standard e r r o r of  fish  27.70 ±1 .45 23.07 ±1.01 21 .77 ±.99 19.78 ±1 .03 24.86 ±.75  7 3  18.74** ±1 .00  Od 8.86 ±.28  9.95 ±.25  9.05 ±.29  3  63d 7.68 ±.10 8.83 ±.50 8.45 ±.17 9.43 ±.19 7.77 ±.21 9.40 ±.63  the mean)  A n a l y s i s of 8 whole f i s h from each chinook s t r a i n at day 0 (2 f i s h per t a n k ) , and 8 whole f i s h from each s t r a i n d i e t group at day 63 (4 f i s h per r e p l i c a t e t a n k ) . Note: P r i o r to day 0, a l l f i s h had been fed only c o n t r o l D i e t 1. Therefore r e s u l t s from the 4 tanks for each s t r a i n were averaged. F i s h sampled w i t h i n any given tank were pooled p r i o r to a n a l y s i s . S-D = Chinook s t r a i n and d i e t combination: Qs = Quesnel; BQ = Big Qualicum; RC = Robertson Creek; D i e t LC = low carbohydrate ( c o n t r o l ) d i e t ; D i e t HC = high carbohydrate d i e t . Dry matter b a s i s . Time on d i e t (days'). * * S i g n i f i c a n t at the a=0.05 l e v e l . * * * S i g n i f i c a n t at the a=0.0l l e v e l . 1  2  3  4  Figure 5: Effect of Dietary Carbohydrate on Body Composition of Chinook Salmon of Three Strains Fed Low or High Carbohydrate Diets for 63 Days. 80-  a  70  Initial 1=1 Day 63 - Low Carta. Diet LTD Day 63 - High Carta. Diet C33  1  **  & c  3  means ± 1 s.e.m.  60  £  Q.  a a o  50  a o  40  a  a OJ  o a  u  LC HC  QUESNEL  LC HC  BIG QUALICUM  Means ± 1 standard error of the mean, N » 2 . * Significant at p < 0.05. * * Significant at p < 0.01. 1  LC HC  ROBERTSON CR.  64 4.2.3.1  Growth i n Terms of P r o t e i n and L i p i d  Instantaneous  p r o t e i n gains  p r o t e i n / d a y , as a percentage instantaneous percentage over the  l i p i d gains  of t o t a l  body p r o t e i n )  trial  f o r the treatment  r a t e s between treatment  fed the high carbohydrate d i e t deposition  groups.  In g e n e r a l ,  demonstrated a lower  was only s i g n i f i c a n t  Robertson Creek chinook  (p<0.05).  IPG  tissue  rate than those fed the c o n t r o l d i e t ,  this difference  groups  are shown in Table 10.  and ILG were c a l c u l a t e d to make comparisons of deposition  and  (ILG, % gain i n l i p i d / d a y , as a  of t o t a l body l i p i d )  63 day feeding  (IPG, % gain i n  lipid  however,  in the a n a l y s i s  There were no  fish  of  significant  e f f e c t s of d i e t on IPG. Final  (day 63)  chinook body weights were a l s o compared  on the b a s i s of average weights of p r o t e i n only and F i g u r e 6 ) . significant 4.2.4  I n d i v i d u a l s t r a i n analyses r e v e a l e d no  e f f e c t s of d i e t  on p r o t e i n weights.  Feed Intake and E f f i c i e n c y The average  (FE,  feed  Indices  intake per f i s h  g wet body weight gain -s- g feed  efficiency energy  (FI),  feed  intake),  r a t i o s of the treatment  efficiency  and energy  (EE, c a r c a s s gross energy gain + feed  intake)  (Table 11  gross  groups over the  day feeding p e r i o d are p r o v i d e d in Table 12.  63  FE and EE  r a t i o s are a l s o i l l u s t r a t e d i n F i g u r e 7. Although there appears to be a trend for decreased intake  i n groups fed the high carbohydrate d i e t  relative  feed to  65  Table  10 - Instantaneous p r o t e i n g a i n (IPG) and instantaneous l i p i d gain (ILG) i n treatment groups over the 63-day feeding p e r i o d .  Strain  Diet  IPG^  1  Quesnel  LC  .696  ±.009  Quesnel  HC  .644  ±.012  B.Qual.  LC  .560  ±.010  B.Qual.  HC  .464  ±.010  Rob.Cr.  LC  .597  ±.038  Rob.Cr.  HC  .535  ±.042  Means ± SEM (standard e r r o r of  ILG 1 .084 ± . 0 8 6 .675  ±.085  1 .221 ± . 0 9 1 .908  ±.049  1 .183 ± . 0 0 6 .486  ±.103  **  the mean)  D i e t LC = low carbohydrate ( c o n t r o l ) d i e t ; D i e t HC = high carbohydrate d i e t . Instantaneous p r o t e i n gain = [ (lnP2 - l n P ^ + T ] x 100 where P2=final weight of p r o t e i n , P ^ i n i t i a l weight of p r o t e i n , and T=time i n days. There were no s i g n i f i c a n t e f f e c t s of d i e t on IPG i n chinook salmon w i t h i n the Quesnel, Big Qualicum or Robertson Creek s t r a i n s . Instantaneous l i p i d gain = [ ( l n L - l n L ) * T ] x 100 where L 2 = f i n a l weight of l i p i d , L i = i n i t i a l weight of l i p i d , and T=time in days. * * S i g n i f i c a n t at the a=0.05 l e v e l . 1  2  3  2  1  66  Table  S-Diet  11  -  Mean p r o t e i n weights of chinook in treatment groups i n i t i a l l y and at the end of the 63 day feeding t r i a l .  Body P r o t e i n Weights  1  Od  63d  Qs - LC  20.7  ±.23  32.0  ±.17  Qs - HC  20.8  ±.10  31.2  ±.09  BQ - LC  16.7  ±.20  23.8  ±.85  BQ - HC  16.4  ±.47  21 .9 + .77  RC - LC  18.1  ±.36  26.3  ±  RC - HC  18.1  ±.42  25.0  ±.21  Means ± SEM (standard e r r o r of the  1.15  mean)  S-Diet = chinook s t r a i n and d i e t combination: Qs = Quesnel; BQ = Big Qualicum; and RC = Robertson Creek. Diet 1 = LC (low carbohydrate) c o n t r o l d i e t ; Diet 2 = HC (high carbohydrate) experimental d i e t . Average weights of body p r o t e i n o n l y . There were no s i g n i f i c a n t e f f e c t s of d i e t on f i n a l (day 63) p r o t e i n weights in chinook salmon w i t h i n the Quesnel, Big Qualicum or Robertson Creek s t r a i n s .  67  Table 12  S-Diet  1  -  Feed intake ( F I ) , feed e f f i c i e n c y (FE) and energy e f f i c i e n c y (EE) r a t i o s for treatment groups at the end of the 63-day feeding p e r i o d .  FI(g)  FE  2  Qs - LC  111.50 ±  2.02  .597  Qs - HC  107.63 ± 1 3 . 8 3  .547  EE  3  ±.006 ±.005  4  .258 ± . 0 0 6  **  .218  ±.037  BQ - LC  70.08 ±  7.52  .545 ± . 0 0 1  BQ - HC  62.46 ±  9.86  .469  RC - LC  86.55 ±  5.85  .576 ± . 0 2 1  .256 ± . 0 1 9  RC - HC  71 .43 ± 1 .34 •  .530 ± . 0 0 5  .181  ±.012  .230 ± . 0 0 1  **  .234 ± . 0 1 6  ±.025  Means ± SEM (standard e r r o r of the mean) S - D i e t = chinook s t r a i n and d i e t combination: Qs = Quesnel; BQ = B i g Qualicum; and RC = Robertson Creek. D i e t LC = low carbohydrate ( c o n t r o l ) d i e t ; D i e t HC = high carbohydrate d i e t . Feed intake = average intake per f i s h (g) over 63 days. Feed e f f i c i e n c y = wet body weight gain (g) -J- feed intake ( g ) . Energy e f f i c i e n c y = c a r c a s s gross energy gain ( k c a l ) feed GE intake ( k c a l ) . Gross energy values used for c a l c u l a t i o n s were: p r o t e i n , 5.65 k c a l / g ; l i p i d , 9.5 k c a l / g ; g e l a t i n i z e d wheat s t a r c h and a - c e l l u l o s e , 4.20 k c a l / g ; and n i t r o g e n - f r e e e x t r a c t , 3.90 k c a l / g (Lloyd et al., 1978). There were no s i g n i f i c a n t e f f e c t s of d i e t on feed intakes or energy e f f i c i e n c y r a t i o s i n chinook salmon w i t h i n the Quesnel, B i g Qualicum or Robertson Creek strains. * * S i g n i f i c a n t at the a=0.05 l e v e l . 1  2 3  4  Figure 6 :  Protein Weights of Chinook Salmon Fed Low or High Carbohydrate Diets for 63 Days.  •Z) Low Carbohydrate Diet — Day 63 IZZ High Carbohydrate Diet - Day 63  means ± 1 s.e.m.  69  Figure 7: Feed Efficiency and Energy Efficiency Ratios of Chinook Salmon of Three Strains Fed Low or High Carbohydrate Diets for 63 Days. 0.750  Diets: QD Low Carbohydrate E23 High Cabohydrate  means ± 1 s.e.m.  11  •o  u c £ o  0.500 •  1= 0.250  21  Quesnel  B. Qualicum  Robertson Cr.  Quesnel  B. Qualicum  Robertson Cr.  0.300  ^ ^  §  0.250  u  § ^  0.200  o cn  |  & 0.150  0.100  Means ± 1 standard error of the mean, N=2. "'Significant at p < 0.05. 1  70  controls, in the  diet  d i d not s i g n i f i c a n t l y  affect  feed consumption  individual strain analyses.  Feed e f f i c i e n c i e s  were g e n e r a l l y  the high carbohydrate d i e t . significant  reductions  reduced in f i s h  I n d i v i d u a l analyses  in feed e f f i c i e n c y  c o n t r o l groups  revealed  in Quesnel and Big  Qualicum chinook fed the h i g h carbohydrate d i e t respective  relative  the high carbohydrate d i e t  ratios relative  were lower  for f i s h  to those of  4.2.5  fed  control  groups i n the Quesnel and Robertson Creek c h i n o o k .  diet  to  (p<0.05).  Mean energy e f f i c i e n c y  individual  fed  s t r a i n a n a l y s e s showed no s i g n i f i c a n t  However,  a f f e c t s of  on E E , due to h i g h v a r i a t i o n among r e p l i c a t e  groups.  Protein U t i l i z a t i o n Protein efficiency  p r o t e i n intake)  {PER, g body weight gain -s- g  and p r o d u c t i v e p r o t e i n values  p r o t e i n gain -r g p r o t e i n and F i g u r e 8.  ratios  In the  were s i g n i f i c a n t l y  intake)  are p r o v i d e d in Table 13  individual strain analyses,  controls  e f f e c t s of d i e t  (p<0.05),  the three chinook  strains.  in these  groups d i d not  with respect to PER.  v a l u e s were not s i g n i f i c a n t l y  relative  to  ( i n accordance with  on feed e f f i c i e n c y  Robertson Creek chinook d i e t significantly  PER values  reduced in Quesnel and B i g Qualicum  chinook fed the high carbohydrate d i e t respective  (PPV, g  the  strains). differ  Productive protein  i n f l u e n c e d by d i e t  in any of  71 Table  13  Strain  -  P r o t e i n e f f i c i e n c y r a t i o s (PER) and p r o d u c t i v e p r o t e i n v a l u e s (PPV) in treatment groups over the 63-day feeding p e r i o d .  Diet  1  PER^  PPV  Quesnel  LC  1 .39 ± . 0 2  Quesnel  HC  1 .27  B.Qual.  LC  B.Qual.  .261  ±.010  .246  ±.030  1 .27 ± . 0 0  .252  ±.002  HC  1 .09 ± . 0 4  .233  ±.010  Rob.Cr.  LC  1 .34 ± . 0 7  .252  ±.012  Rob.Cr.  HC  1.15  ±.07  .246  ±.012  ±.02  Means ± SEM (standard e r r o r of  **  the mean)  D i e t LC = low carbohydrate ( c o n t r o l ) d i e t ; D i e t HC = high carbohydrate d i e t . P r o t e i n e f f i c i e n c y r a t i o = body weight gain (g) * p r o t e i n intake (g). P r o d u c t i v e p r o t e i n value = p r o t e i n gain (g) + p r o t e i n intake (g). There were no s i g n i f i c a n t e f f e c t s of d i e t on PPV in chinook salmon of the Quesnel, B i g Qualicum or Robertson Creek s t r a i n s . * * S i g n i f i c a n t at the a=0.05 l e v e l . 1  2  3  72  Figure 8 :  2.00  % I  Protein Efficiency Ratios and Productive Protein Values of Chinook Salmon of Three Strains Fed Low or High Carbohydrate Diets for 63 Days. Diets: CZ3 Low Carbohydrate 7Z2 High Cabohydrate  1.50 +  I  g  1.00-•  1  *  0.50 +  0.00  V?  'A  Quesnel  B. Qualicum  0.40 _  0.35  |  |  0.30 +  f  |  0.25 +  % 8 £ £ |  0.20 +  §> 0.15 +  eI °- i  0.10+ 0.05 + 0.00  Robertson Cr.  means ± 1 s.e.m  0 2 Quesnel  P7  id  B. Qualicum  Means ± 1 standard error of the mean. N=2. •Significant at p < 0.05. 1  means ± 1 s.e.m.  Robertson Cr.  73  4.2.6  L i v e r Glycogen and Hepatosomatic Index Percent l i v e r glycogen l e v e l s  indices Table  (LG) and hepatosomatic  (HSI, l i v e r weight:body weight  ratios)  are given in  14 for day 0, day 63 (end of experimental  feeding  p e r i o d ) and day 84 (end of 21 day feed withdrawal p e r i o d ) . Additionally, of  day 63 HSI were c a l c u l a t e d with the  l i v e r glycogen  (HSI-G),  treatments on l i v e r weight l i v e r glycogen l e v e l . graphically  in Figure  to examine the e f f e c t  exclusion of  the  independent of a l t e r a t i o n s  in  L i v e r glycogen l e v e l s are presented 9.  A r c s i n e t r a n s f o r m a t i o n was c a r r i e d out on these data p r i o r to analyses  of v a r i a n c e .  Individual strain  analyses  r e v e a l e d higher HSI i n chinook of the Quesnel and Robertson Creek s t r a i n s respective  fed high carbohydrate d i e t  c o n t r o l groups.  no d i e t a r y i n f l u e n c e s  B i g Qualicum chinook e x h i b i t e d  on HSI.  A n a l y z i n g HSI with l i v e r  glycogen excluded (HSI-G) e s s e n t i a l l y results, (a=0.05).  (p<0.01), than i n  d i d not change  the  but only the s i g n i f i c a n c e p r o b a b i l i t y l e v e l of a At 21 days post  experiment),  feed withdrawal (day 84 of  a l l HSI had d e c l i n e d to  values and no s i g n i f i c a n t d i f f e r e n c e s  (or below)  initial  were found between  d i e t a r y treatment groups. L i v e r glycogen l e v e l s were s i g n i f i c a n t l y e l e v a t e d the groups fed high carbohydrate d i e t Robertson Creek chinook (p<0.05)  in the Quesnel and  i n the i n d i v i d u a l  Percent l i v e r glycogen l e v e l s ranged from 3.2 Quesnel chinook, from 2 to 7.5  in  to  analyses.  11.3  in  i n B i g Qualicum chinook, and  74 Table  S-D  1  14  -  Mean percent l i v e r glycogen (LG) l e v e l s and hepatosomatic i n d i c e s (HSI) i n treatment groups ( i n i t i a l l y , d u r i n g feeding and 21 days post feed w i t h d r a w a l ) .  % L i v e r Glycogen  Hepatosomatic Index^  Od  63d  84d  Qs-LC  1 .34 ±.53  2.18 ±.22  0.73 ±.09  Qs-HC  1 .51 ±.26  BQ-LC  1 .64 ±.47  BQ-HC  4  Od  63d  84d  HSI-G 4  63d  1 .36 ±.05  1 .48 ±.05  ±.45  1 .33 ±.06  2.08** 1 .26 ±.04 ±.09  .91** ±.05  1.13 ±.09  0.41 ±.12  1 .59 ±.10  1 .98 ±.05  1 .29 ±.03  1 .94 ±.03  1 .36 ±.21  3.59 ±1 .06  0.24 ±.03  1 .67 ±.01  1 .95 ±.08  1 .30 ±.02  1 .86 ±.05  RC-LC  1 .42 ±.23  1 .40 ±.01  0.48 ±.07  1 .37 ±.03  1 .62 ±.01  1 .35 ±.09  1 .59 ±.01  RC-HC  1 .62 ±.02  5.25*** 0.39 ±.48 ±.02  1 .44 ±.06  1.94** 1 .43 ±.02 ±.12  7.32 ±.95  * * * 0.84  1 .24 ±.12  1 .46 ±.05  .85** ±.03  Means ± SEM (standard e r r o r of the mean) S-D = Chinook s t r a i n and d i e t combination: Qs = Quesnel; BQ = Big Qualicum; and RC = Robertson Creek D i e t LC = low carbohydrate ( c o n t r o l ) d i e t ; D i e t HC = high carbohydrate d i e t . HSI = L i v e r weight:body weight r a t i o . HSI-G = HSI r e c a l c u l a t e d with l i v e r glycogen weight excluded. Day 84 was the end of a 21 day feed withdrawal p e r i o d (from day 63 to day 84). There were no s i g n i f i c a n t d i f f e r e n c e s between d i e t groups i n HSI or %LG l e v e l s i n chinook salmon 21 days a f t e r feed withdrawal w i t h i n the Quesnel, B i g Qualicum or Robertson Creek s t r a i n s . * * S i g n i f i c a n t at the a=0.05 l e v e l . * * * S i g n i f i c a n t at the a=0.0l l e v e l . 1  2  3  4  Figure 9  Effect of Dietary Carbohydrate on Liver Glycogen Level in Chinook Salmon of Three Strains: During Feeding and subsequent to feed withdrawal.  10 987c  Q> O  O  o  STRAIN: Low Carb. High Carb. Quesnel LZ3 B. Qualicum LZZ1 rxzi Robertson Cr \ZS  means ± 1 s.e.m  1  6543-  >  21 0 63 Time (days)  ^ Means ± 1 standard error of the mean, N—2. *Significant at p < 0.01.  84 ( 21 days after feed withdrawal )  76  from 2.2  to 8.9  in Robertson Creek chinook fed the h i g h  carbohydrate d i e t .  After  21 days of  decreased to b a s a l l e v e l s , between treatments groups analyses of 4.2.9  feed withdrawal, a l l LG  with no s i g n i f i c a n t ( i n the o v e r a l l or  differences  individual  variance).  Mortality Mortality  in chinook of the Quesnel and Robertson Creek  s t r a i n s was n e g l i g i b l e  i n a l l groups.  experienced m o r t a l i t y p r i m a r i l y growth t r i a l  (Table 15),  i n the l a t t e r p a r t of  and 11 m o r t a l i t i e s  groups fed the high carbohydrate d i e t other).  the  with a t o t a l of 6 m o r t a l i t i e s  c o n t r o l groups (3 per r e p l i c a t e ) ,  and 5 i n the  Big Qualicum chinook  (6 i n one  in  in  replicate,  77  Table 15  -  Strain  T o t a l m o r t a l i t i e s i n treatment 63-day feeding p e r i o d .  Diet  1  Total  Mortalities  groups over  (NO. Of f i s h )  Quesnel  LC  1  ( 53 )  Quesnel  HC  0  ( 52 )  Big Qualicum  LC  6  ( 60 )  Big Qualicum  HC  1 1  ( 60 )  Robertson C r .  LC  1  ( 60 )  Robertson C r .  HC  2  ( 60 )  D i e t LC = low carbohydrate ( c o n t r o l ) D i e t HC = high carbohydrate d i e t .  diet;  the  78  4.3  DISCUSSION  4.3.1  General In t h i s  examined,  study,  chinook salmon of the d i f f e r e n t  differed significantly  i n mean body weights, and  degrees of v a r i a t i o n around the means. in growth p o t e n t i a l ,  Genetic  time of s m o l t i f i c a t i o n  differences  and behaviour  (Withler et  al.,  and L a r k i n ,  1986), as w e l l as the extended h o l d i n g p e r i o d  and r e s t r i c t e d differences.  1987;  strains  feeding  C l a r k e and Shelbourn, 1985;  c o n t r i b u t e d to these  On the other hand, i n i t i a l  Taylor  size  body weights of  the  four experimental groups w i t h i n each chinook s t r a i n , d i d not differ  significantly.  As d i f f e r e n c e s  in s i z e were a s s o c i a t e d  chinook s t r a i n s ,  with  different  the two v a r i a b l e s were confounded, and s i z e  c o u l d not be a d j u s t e d or c o r r e c t e d for i n the analyses.  experimental  Therefore chinook of each s t r a i n were assessed  i n d i v i d u a l l y for carbohydrate u t i l i z a t i o n .  Any attempted  comparison between chinook of the d i f f e r e n t  strains  study must be made i n l i g h t of the  fact  that  in  this  body weight  is  c o r r e l a t e d with metabolism. Data from Shuswap chinook were not due to s e r i o u s  i n c l u d e d in analyses  b a c t e r i a l kidney d i s e a s e outbreak in a l l  experimental groups of t h i s  strain.  79 4.3.2  Growth Rates Although the trends  i n body weight suggest a r e d u c t i o n  in growth i n chinook fed the high carbohydrate differences  were not s i g n i f i c a n t  diet,  w i t h i n Quesnel, Big  Qualicum or Robertson Creek chinook s t r a i n s .  This finding  was confirmed in the examination of body weight gains the 63 day feeding a l t e r e d by d i e t  trial,  in the three  More r e v e a l i n g , growth r a t e s  which were not  over  significantly  strains.  however,  are the analyses of  specific  (SGR, percent gain i n body weight per day)  d u r i n g the three  21-day  4 clearly illustrates  i n t e r v a l s between weighings.  a decline,  growth i n t e r v a l , i n s p e c i f i c  growth r a t e s of  c a r b o h y d r a t e - f e d chinook r e l a t i v e c o n t r o l groups.  p a r t i c u l a r l y i n the  second  high  to those of  Following this decline,  Figure  respective  SGR improved i n the  t h i r d growth i n t e r v a l , and were comparable to those of c o n t r o l groups. curves d i v e r g e then e x h i b i t  Figure 3 also depicts in the  t r e n d , as growth  and second growth  intervals,  s i m i l a r slopes in the t h i r d growth i n t e r v a l .  This indicates  that an a d a p t a t i o n response  carbohydrate d i e t growth t r i a l .  first  this  to the high  o c c u r r e d w i t h i n the 63 day d u r a t i o n of  The narrower margin of d i f f e r e n c e  in f i n a l  mean weights between groups fed the high and low carbohydrate d i e t s i n Quesnel chinook suggests a s t r a i n difference  i n response  to the high carbohydrate  diet.  the  80  4.3.3  Carcass Composition Figure 5 i l l u s t r a t e s  the changes i n c a r c a s s  of chinook over the 63-day  feeding  trial.  Initial  composition data are shown for comparative F i s h fed the high carbohydrate d i e t body f a t ,  composition carcass  purposes.  g e n e r a l l y had lower  but higher body p r o t e i n and ash than those  control diet.  S i m i l a r responses to high carbohydrate d i e t s  have a l s o been demonstrated (Beamish et  al.,  and A u s t r e n g ,  fed  1986;  1981).  in s t u d i e s on rainbow t r o u t  H i l t o n and A t k i n s o n , 1982; The d i e t a r y e f f e c t s were  Refstie  significant  in B i g Qualicum chinook for % c a r c a s s p r o t e i n , and i n Robertson Creek chinook for both % c a r c a s s p r o t e i n and % carcass  fat.  Chinook fed the c o n t r o l d i e t  higher but n o n - s i g n i f i c a n t  a l s o had s l i g h t l y  % c a r c a s s dry matter l e v e l s than  noted for the high c a r b o h y d r a t e - f e d groups. 4.3.3.1  Growth i n Terms of Carcass Composition  In view of the d i f f e r e n c e s  in carcass composition,  body  weights were re-examined on the b a s i s of p r o t e i n weight, shown in F i g u r e 6. significantly amount of  as  F i n a l p r o t e i n weights were not  affected  by d i e t ,  i n d i c a t i n g that a s i m i l a r  ( t o t a l ) p r o t e i n was l a i d down when chinook were  fed to s a t i a t i o n  on e i t h e r  the low or high carbohydrate  diets. Instantaneous  p r o t e i n and l i p i d gains  (Table 10) were  c a l c u l a t e d to d e s c r i b e the d a i l y r a t e s of p r o t e i n and fat deposition  (percent  fat gain per day and p e r c e n t  protein  81  gain per day, r e s p e c t i v e l y )  relative  to i n i t i a l  levels.  These i n d i c e s were c a l c u l a t e d i n the same manner as growth r a t e , final  using the n a t u r a l logarithms of  p r o t e i n weights or f a t weights.  gains were c o n s i s t e n t l y carbohydrate d i e t significant  lower  exhibited  protein  i n f i s h fed the high  but d i e t a r y d i f f e r e n c e s  were not  i n any one of the three chinook s t r a i n s  tested,  In the  s t r a i n analyses only Robertson Creek chinook significantly  carbohydrate d i e t .  lower IFG i n groups fed the high  B i g Qualicum chinook,  d i s p l a y e d the g r e a t e s t r e l a t i v e narrowest margin of d i f f e r e n c e 4.3.4  i n i t i a l and  Instantaneous  as confirmed by the f i n a l p r o t e i n weights. individual  specific  fat  on the other hand,  gains o v e r a l l , and the  between d i e t  groups.  Feed Intake and Feeding Response Feed intake was not s i g n i f i c a n t l y  affected  although feed consumption was g e n e r a l l y the h i g h carbohydrate d i e t . overall  lower for f i s h  This p a r t i a l l y explains  r e d u c t i o n in growth on the high carbohydrate  In Robertson Creek chinook, intakes  by d i e t ,  between d i e t  two s t r a i n s ,  the margin of d i f f e r e n c e  groups was g r e a t e r  the diet. in  feed  other  and both r e p l i c a t e groups fed the high  carbohydrate d i e t  exhibited consistently  than c o n t r o l groups.  Nonetheless,  lower feed  intakes  high v a r i a b i l i t y between  r e p l i c a t e groups obscured any p o t e n t i a l differences.  than i n the  fed  significant  Feed intake has been shown to d e c l i n e  in  82  rainbow t r o u t the  fed high l e v e l s of d i g e s t i b l e  form of extruded s t a r c h Feeding responses of  diet  d i f f e r e d noticeably  carbohydrate i n  ( H i l t o n and S l i n g e r ,  1983).  f i s h fed the high carbohydrate from those of the c o n t r o l groups,  d u r i n g the course of the feeding  trial.  Observations  i n c l u d e d a reduced enthusiasm for the h i g h carbohydrate accompanied by increased s p i t t i n g of  feed p e l l e t s  satiation  was approached.  difficult  to determine when s a t i a t i o n  groups.  Interestingly,  It was consequently  regard.  response  between d i e t  groups were d i f f i c u l t  more  strains did  While Robertson Creek  chinook e x h i b i t e d the most conspicuous feeding  as  was reached i n these  chinook from d i f f e r e n t  not behave e q u a l l y i n t h i s  differences  in  groups, Quesnel chinook  to d i s t i n g u i s h .  In f a c t ,  the  intake of one of the high carbohydrate r e p l i c a t e s  diet  feed  of Quesnel  chinook a c t u a l l y exceeded that of the c o n t r o l groups. a d d i t i o n to d i e t a r y induced d i f f e r e n c e s , c o u l d be d i s t i n g u i s h e d and e x c i t a b i l i t y . satiation 4.3.5  chinook  i n terms of general  Thus accurate  feed  feeding  In  strains  feeding  behaviour  of a l l groups  to  proved d i f f i c u l t .  Feed and Energy E f f i c i e n c y The observed d i f f e r e n c e s  weight gain -f feed strains  intake)  i n feed e f f i c i e n c i e s  among the d i f f e r e n t  (Figure 7) were l i k e l y a s s o c i a t e d  differences  i n body weight.  and r a t e of growth.  For example,  body  chinook  with the  Fish size affects  (wet  strain  metabolism  smaller f i s h grow  faster  83  on a percent  body weight b a s i s but r e q u i r e more energy per  u n i t weight for metabolism than l a r g e r species.  Thus lower  expected in smaller requirement  feed e f f i c i e n c y f i s h due to the  relative  to body weight  f i s h of the same  r a t i o s would be increased (Halver,  F i s h fed the h i g h carbohydrate d i e t lower  feed e f f i c i e n c y  ratios  Qualicum chinook s t r a i n s .  maintenance 1989).  had  significantly  in both Quesnel and Big  Energy e f f i c i e n c y  means e x h i b i t e d  a downward trend in h i g h - c a r b o h y d r a t e fed chinook of Quesnel and Robertson Creek s t r a i n s ,  although data were  h i g h l y v a r i a b l e , and the d i e t a r y d i f f e r e n c e s (Figure 7 ) .  This trend indicates  h i g h carbohydrate d i e t that body.  non-significant  that a greater q u a n t i t y of  was r e q u i r e d to achieve  l e s s of the energy These r e d u c t i o n s  the  from t h i s d i e t  g a i n , and  was r e t a i n e d i n  in feed and energy  the  efficiency  suggest poorer u t i l i z a t i o n of the c a r b o h y d r a t e .  Feed and  energy e f f i c i e n c i e s  due to  loss,  especially  problems s u f f e r e d of  may have been underestimated  among high c a r b o h y d r a t e - f e d groups.  Health  by Big Qualicum chinook in the l a t t e r  the growth t r i a l ,  l i k e l y affected  feed  feeding  efficiency.  In Quesnel and Robertson Creek chinook,  on average,  part  26%  of d i e t a r y gross energy was r e t a i n e d in the c a r c a s s e s of control-fed  fish.  T h i s compares to an average  of 22% for  Quesnel chinook and 18% for Robertson Creek chinook groups fed the high carbohydrate d i e t . f i s h r e t a i n e d l e s s of  in  Thus i t appears  the carbohydrate energy,  that  whereas those  84  fed the c o n t r o l d i e t more r e a d i l y converted excess energy adipose  to  tissue.  The d i e t s employed i n t h i s study were formulated to be i s o n i t r o g e n o u s and ( t h e o r e t i c a l l y )  isoenergetic  (Table 2 ) .  The m e t a b o l i z a b l e energy values used to estimate a v a i l a b l e energy of the two t e s t d i e t s were; 4.5 kcal/g l i p i d ,  3.8  1986), and 3.0  kcal/g protein,  k c a l / g animal s t a r c h (Beamish et  8.5  al.,  k c a l / g g e l a t i n i z e d wheat s t a r c h (assuming a  75% d i g e s t i b i l i t y )  (Singh and Nose,  carbohydrate d i e t had 25% of  1967).  The high  i t s ME in the form of  carbohydrate and 19% i n the form of  fat  (30 % g e l a t i n i z e d  wheat s t a r c h and 8% f i s h o i l , r e s p e c t i v e l y ) . the c o n t r o l d i e t had 44% of  By c o n t r a s t ,  i t s ME i n the form of  f a t and a  n e g l i g i b l e amount of ME (1.6%) from c a r b o h y d r a t e .  However,  H i l t o n et  al .  (1987),  retention  i n rainbow t r o u t ,  l i t e r a t u r e ME values Interesting,  i n t h e i r examination of c a r c a s s r e p o r t e d o v e r e s t i m a t i o n of  for s t a r c h and g l u c o s e .  B i g Qualicum chinook fed the high  carbohydrate d i e t d e p o s i t e d more l i p i d initial  lipid  levels  margin of d i f f e r e n c e  (see  I L G , Table  r e l a t i v e to t h e i r  10),  narrowing the  i n % c a r c a s s fat between d i e t  groups,  in comparison to the other s t r a i n s .  This is  the higher average energy e f f i c i e n c y  r a t i o of the h i g h  reflected  c a r b o h y d r a t e - f e d groups (23%), which i s s i m i l a r to average value for c o n t r o l groups. inconsistencies 'apparent'  energy  Nevertheless,  among r e p l i c a t e groups, none of  differences  were s t a t i s t i c a l l y  the  due to the  significant.  in  85  4.3.6  U t i l i z a t i o n of  Protein  Protein efficiency intake)  ratios  were s i g n i f i c a n t l y  (wet  weight gain * p r o t e i n  reduced in both Quesnel and  Robertson Creek chinook fed the high carbohydrate (Figure 8 ) ,  c o n s i s t e n t with the d i f f e r e n c e s  efficiencies.  in feed  However, PER v a l u e s alone are of  usefulness in assessing protein u t i l i z a t i o n , take  i n t o account  the nature of  P r o d u c t i v e p r o t e i n values intake), by d i e t final  limited  as they f a i l  ( p r o t e i n gain  protein affected  T h i s i s confirmed by the a n a l y s e s of  p r o t e i n weights and instantaneous p r o t e i n g a i n s ,  a l s o r e v e a l e d no s i g n i f i c a n t groups,  differences  w i t h i n each s t r a i n , a f t e r  The s l i g h t  but n o n - s i g n i f i c a n t  between  diet  63 days on the t e s t  decline  in f i n a l  which  diets.  protein  weights and IPG of groups fed the high carbohydrate d i e t probably due to the  to  the weight g a i n .  on the other hand, were not s i g n i f i c a n t l y (Figure 8 ) .  diet  (non-significant)  intake and hence growth,  was  r e d u c t i o n i n feed  rather than a d e c l i n e  in p r o t e i n  utilization. Importantly, carbohydrate d i e t  these r e s u l t s  indicate  that  reported s i m i l a r findings 1985;  high  was as e f f e c t i v e as the c o n t r o l d i e t  sparing d i e t a r y p r o t e i n for p r o t e i n s y n t h e s i s .  Oliva Teles,  the  in rainbow t r o u t  Pieper and P f e f f e r ,  in  Others have  (Kaushik and de  1980a,  1980b).  86  Greater f l u i d i t y  i n the  feces of  f i s h fed the h i g h  carbohydrate d i e t  relative  to c o n t r o l groups was observed  during d i s s e c t i o n  of  i n d i c a t i n g an osmotic  4.3.7  Liver  fish,  Effects  Percent l i v e r glycogen  levels  (Figure 9) were  in a l l groups fed the high carbohydrate d i e t , significant (p<0.0l). glycogen  effect.  elevated  but t h i s  was  only for Quesnel and Robertson Creek chinook Quesnel chinook e x h i b i t e d the h i g h e s t  liver  l e v e l s among the three chinook s t r a i n s t e s t e d ,  i n d i v i d u a l values  ranging from 3.2  to  11.3  e x p l a i n e d by the a p p a r e n t l y b e t t e r  feed  rate on the high carbohydrate d i e t  in t h i s  greater elevation  of h e p a t i c  any d e t r i m e n t a l e f f e c t  glycogen  on f e e d i n g ,  %.  with  T h i s may be  intake and growth strain.  The  d i d not appear to have  h e a l t h or growth i n  Quesnel chinook over the nine week experimental p e r i o d . lower l i v e r glycogen deposition  liver  fat  i n Big Qualicum chinook fed high carbohydrate  d i e t may i n d i c a t e strain.  l e v e l s and higher r e l a t i v e  The  some d i f f e r e n c e s  in metabolism in  There were no obvious e f f e c t s of d i e t  this  on chinook  colour. While HSI were e l e v a t e d  carbohydrate d i e t  relative  to c o n t r o l groups i n both Quesnel  and Robertson Creek s t r a i n s , elevation  i n both d i e t  differences  in chinook fed the high  B i g Qualicum chinook  groups, and no  between d i e t  groups.  exhibited  significant  Refstie  and Austreng  87  (1981) found a s i g n i f i c a n t and d i e t  i n t e r a c t i o n between t r o u t  family  for HSI.  Analyses of HSI r e c a l c u l a t e d with the weight of glycogen  subtracted  statistical of  outcome,  significant  therefore  (HSI-G) had l i t t l e  effect  hepatic  on the  changing only the a p r o b a b i l i t y l e v e l s  differences.  Glycogen d e p o s i t i o n  only p a r t i a l l y r e s p o n s i b l e  was  for i n c r e a s e s  in l i v e r  weight. Feed withdrawal l e d to a d e c l i n e initial  values  i n HSI t o ,  i n each chinook s t r a i n .  Liver  or  below,  glycogen  l e v e l s had d e c l i n e d to l e s s than 1% i n a l l groups 21 days after  feed withdrawal.  l i v e r glycogen  In rainbow t r o u t s t u d i e s ,  l e v e l s and hepatosomatic  the h i g h carbohydrate d i e t , 12 days of Hilton, 4.3.8  indices  elevated in f i s h  d e c l i n e d to normal v a l u e s  feed withdrawal ( H i c k l i n g and March,  fed  after  1982;  1982). Mortality  H e a l t h problems arose d u r i n g the study.  in two of the chinook  strains  Shuswap chinook u n f o r t u n a t e l y had to be  e l i m i n a t e d from experimental a n a l y s e s due to an outbreak of B a c t e r i a l Kidney D i s e a s e . some m o r t a l i t y i n the although most  B i g Qualicum chinook  latter  t h i r d of the growth  suffered trial,  f i s h appeared vigorous during the study and  those sampled had no obvious s i g n s of  disease.  Higher m o r t a l i t y was observed i n Big Qualicum chinook fed the high carbohydrate d i e t  i n comparison to c o n t r o l  88  groups.  T h i s may r e f l e c t  carbohydrate-fed  a reduced a b i l i t y of  f i s h to defend  High carbohydrate f e e d i n g , glycogen,  the body a g a i n s t  and hence e l e v a t e d  has been shown to cause s u b l e t h a l  rainbow t r o u t , and H i l t o n , toxicities.  and selenium  effects  1982;  trout.  strains,  al.,  1948)  or have no e f f e c t  Austreng et  al.,  In the present study,  mortality  in (Dixon  1983)  high d i e t a r y l e v e l s of  d i g e s t i b l e carbohydrate have been shown to  Atkinson,  liver  ( H i l t o n and Hodson,  In growth t r i a l s ,  et  infection.  i n c l u d i n g reduced r e s i s t e n c e to copper  1985)  (Phillips  high  1977)  increase on ( H i l t o n and  mortality  diet exhibited  i n rainbow  no e f f e c t  on  in chinook of the Quesnel and Robertson Creek which remained healthy  throughout  the growth  trial.  89  4.4  CONCLUSIONS  (Experiment  1)  The r e s u l t s of t h i s study are able to t o l e r a t e gelatinized  indicate  digestible  carbohydrate i n the  wheat s t a r c h at a d i e t a r y l e v e l  Growth r a t e s i n d i c a t e d an a d a p t a t i o n carbohydrate w i t h i n the of  that chinook  of  300  response to  significant  depressed  high in carbohydrate l e d  changes in c a r c a s s  composition,  and decreased  Feed intake was s l i g h t l y  strains.  to  including  fat  relative  but not  significantly  Feed e f f i c i e n c y  and energy  efficiency  e x h i b i t e d a downward t r e n d , although the l a t t e r significant,  due to high r e p l i c a t e  l e s s energy  carcass  deposition.  variability.  significantly  the  i n d i c a t i n g a protein sparing effect of the  i n i t i a l body weights of chinook  between the  fat  strains  tested,  carbohydrate u t i l i z a t i o n . strain differences.  the  Some o b s e r v a t i o n s For example,  of  lipid. differed  r e s u l t s could  not be a s c r i b e d p u r e l y to g e n e t i c d i f f e r e n c e s  inherent  appears  p r o t e i n u t i l i z a t i o n was not  the carbohydrate comparable to that the  It  as confirmed by the lower c a r c a s s  Importantly,  affected,  Because  was not  from carbohydrate was r e t a i n e d in  than from f a t ,  adversely  to  in high carbohydrate groups and reduced a p p e t i t e  c o u l d be observed.  that  g/kg.  63 day experimental p e r i o d in each  i n c r e a s e d p r o t e i n and a s h , controls.  form of  the  the Quesnel, B i g Qualicum and Robertson Creek  Consumption of a d i e t  salmon  in however  the  suggest  superior  growth r a t e s of Quesnel chinook fed high carbohydrate  90  relative  to c o n t r o l s ,  carbohydrate  and b e t t e r a p p e t i t e for the  diet.  L i v e r glycogen significantly  l e v e l s and hepatosomatic  showed no d i f f e r e n c e s  concentrations the other  chinook,  the  i n hepatosomatic  indices  glycogen  in the high carbohydrate groups than chinook  strains.  higher r e l a t i v e difference  were  Big Qualicum chinook,  between the two d i e t s and had lower l i v e r  of  indices  e l e v a t e d in high c a r b o h y d r a t e - f e d f i s h of  Quesnel and Robertson Creek s t r a i n s . however,  high  T h i s combined with the o b s e r v a t i o n  body fat  i n these groups suggests a  i n carbohydrate u t i l i z a t i o n by Big Qualicum  compared with the other  higher energy e f f i c i e n c y  strains  63-day  feeding  c o n s i s t e n t with the lower l i v e r glycogen concentrations.  Quesnel chinook,  h i g h e s t l i v e r glycogen  in the  study.  The  d i s p l a y e d by high carbohydrate  groups of t h i s s t r a i n d u r i n g the  period  is  and higher body  concentrations,  yet  the best  feeding  diet,  i n d i c a t i n g that d u r i n g the time frame of the  experiment,  l i v e r glycogen  concentrations  by f i s h of t h i s  fat  on the other hand, had the  and growth response on the high carbohydrate  tolerated  of  of as high as  11.3% were  strain.  The h i g h carbohydrate had no e f f e c t Quesnel and Robertson Creek chinook.  on m o r t a l i t y  However,  in  higher  m o r t a l i t y i n Big Qualicum chinook fed the high carbohydrate diet,  may i n d i c a t e a reduced c a p a c i t y  (assuming  that  to combat  the cause of m o r t a l i t y was an  infection  infection).  91  Longer term feeding  trials,  i n c l u d i n g examination of  key enzymes i n v o l v e d in carbohydrate metabolism  for  a d a p t a t i o n response,  research.  are recommended for future  92 5  EXPERIMENT 2  5.1  Part i )  5.1.1  O r a l Glucose T o l e r a n c e in S e l e c t e d B. C . Chinook Salmon.  MATERIALS AND METHODS  5.1.1.1  Tank P r e p a r a t i o n and F i s h A l l o c a t i o n  A s e r i e s of glucose t o l e r a n c e 4 of  S t r a i n s of  t r i a l s was c a r r i e d out on  the 7 remaining chinook stocks which had been h e l d i n  the 2.5 m diameter 1988.  6000 1) outdoor tanks s i n c e March  The seawater flow  tank, DO ranged from 7.0  r a t e was approximately 35 1/min per to  10.0  ppt,  s a l i n i t y v a r i e d from  26 to 30 ppt and temperature d e c l i n e d from 10°C to over the course of the t r i a l s . t e s t i n g a given s t o c k ,  Two to 3 weeks before  The seawater supply was  through a f i n e nylon mesh to prevent any small organisms In  from e n t e r i n g the  minimize s t r e s s , different after  test,  (3,  passed edible  6,  12,  18,  sampled at  In order to  f i s h were h e l d i n separate  glucose a d m i n i s t r a t i o n ) .  included.  blood i s  a glucose c h a l l e n g e .  sampling times  8'  tank.  the glucose t o l e r a n c e  v a r i o u s times a f t e r  8.3°C  the f i s h were moved to a c l e a n  diameter h o l d i n g tank.  16,  24,  tanks for  the  30 and 36 hours  A sham group was a l s o  Two banks of e i g h t 200 1 f i b e r g l a s s  tanks,  each  with a nylon mesh cover and a seawater flow r a t e of 5 1 / m i n , were set  up i n the  indoor f a c i l i t y .  The outer 2/3  of  tank top was covered with opaque m a t e r i a l to minimize  each  93  disturbance.  Water temperature was recorded throughout  each  test. 5.1.1.3  Glucose A d m i n i s t r a t i o n : P r e l i m i n a r y T r i a l  To determine the best method of d e l i v e r i n g glucose the f i s h ,  to  a p r e l i m i n a r y comparative t r i a l was conducted on  October 26,  1988. Glucose was a d m i n i s t e r e d o r a l l y by  i n t u b a t i o n of e i t h e r  a) g e l a t i n capsules  f i l l e d with glucose  powder or b) a concentrated glucose s o l u t i o n .  Eighteen  Robertson Creek chinook were d i v i d e d among s i x  200 1 indoor  tanks,  3 f i s h per tank.  The flow  rate of seawater was 5  1/min/tank and water temperature was 10.1 ° C . Treatment a ) : anesthetized 0.1  g).  weight  One group of  in 2-phenoxyethanol  3 f i s h was c a p t u r e d , (0.35  ml/1)  singly  then weighed  The glucose dose was a d j u s t e d on the b a s i s of (167 mg glucose/100 g body w e i g h t ) .  a d m i n i s t e r e d v i a 3 or 4 g e l a t i n  capsules  i n t o the  fish's  stomach.  ( s i z e 2)  The f i s h were  recovery from a n e s t h e s i a .  procedure was c a r r i e d out on the Treatment b ) :  directly  glucose dose and  exact time of a d m i n i s t r a t i o n were r e c o r d e d . returned to t h e i r tank a f t e r  pre-filled  were d e l i v e r e d  a p p l i c a t o r tube  F i s h weight,  fish  The dose was  with glucose monohydrate powder. The capsules through a 7 mm diameter p l a s t i c  first  3 groups of  For the remaining 3 groups of  the method of glucose d e l i v e r y d i f f e r e d . intubated d i r e c t l y i n t o the f i s h ' s  (±  This  fish. fish,  only  Glucose was  stomach through 2 mm  diameter cannula tubing attached to a 100 ul g l a s s  syringe.  94  The s y r i n g e was f i l l e d to a p p r o p r i a t e volume with 80% glucose  solution  (80 g glucose  water),  c a l c u l a t e d a c c o r d i n g to the f o l l o w i n g e q u a t i o n :  s o l u t i o n = .167 g/100 (cone,  of glucose  glucose  monohydrate/100 ml d i s t i l l e d  g (glucose dosage r a t e )  solution)  -f .909165 (cone,  4 and 7 hours  Plasma glucose  in  shown i n F i g u r e capsules  10.  5.2.6.  e x h i b i t e d a slower e s c a l a t i o n  indicates  fish  The r e s u l t s  are  in g e l a t i n  of plasma  in solution  glucose,  form.  This  that the time r e q u i r e d for capsule breakdown  causes a s u b s t a n t i a l delay i n glucose glucose  tolerance  groups of  F i s h a d m i n i s t e r e d glucose  r e l a t i v e to those given glucose  after  l e v e l s were determined  a c c o r d i n g to the p r o t o c o l i n s e c t i o n  uptake.  was a d m i n i s t e r e d i n s o l u t i o n  t e s t s to  For t h i s for a l l  f i s h may r e g u r g i t a t e the glucose  was the  possibility  solution.  For t e s t i n g  purposes o n l y , a b r i g h t red food dye was added to s o l u t i o n to make i t v i s i b l e i n t o the stomachs of s e v e r a l  i n water. fish.  d u r i n g recovery from a n a e s t h e s i a , thereafter,  glucose  follow.  Another p o i n t of c o n c e r n , however, that  of glucose  a d m i n i s t r a t i o n from the 3 r e s p e c t i v e  in each treatment.  reason,  -f .8 g/ml  monohydrate powder) x body weight.  Blood samples were taken at 2, glucose  mis  It was then  the intubated  O b s e r v a t i o n of the  fish  and for 20 minutes  d i d not r e v e a l any evidence of  regurgitation.  Figure 10 :  Preliminary Comparison Trial of Glucose Administration: Capsules VS Solution  Glucose Administration: 400 + In Solution • • In Gelatin Capsules o —  o  300 +  200-  100-Means ± standard error of the mean, N=3.  0 ( before )  + 2  + 3  + 4  + 5  + 6  7  8  Hours After Glucose Administration cn  96  5.1.1.4  Glucose Tolerance T r i a l s  T h i s s e r i e s of glucose t o l e r a n c e between November 17,  t e s t s was conducted  1988 and December 8,  1988.  Harrison,  Robertson Creek, N i t i n a t and Big Qualicum chinook were used i n t h i s p a r t of the study.  Each s t r a i n group was  t e s t e d i n d u p l i c a t e t r i a l s h e l d approximately All  stocks  1 week a p a r t .  were fed a dry commercial r a t i o n u n t i l commencement  the glucose t o l e r a n c e  trial.  of  Water temperature ranged from  8 . 6 ° C to 1 0 ° C . Before each t r i a l , hours,  feed was w i t h h e l d for 63.5  to ensure that a l l f i s h were i n the  state.  The a p p r o p r i a t e number of  to 69  post-absorptive  f i s h was captured from the  h o l d i n g tank and d i s t r i b u t e d among four 30 1 buckets of aerated seawater. i d e a l number of trials,  As some stocks c o n t a i n e d l e s s than  fish,  adjustments  had to be made.  c e r t a i n t e s t p o i n t s were e x c l u d e d ,  number used i n any t r i a l  was 36 f i s h  f i s h per sampling time.  At the s t a r t  were a n e s t h e t i z e d  i n 2-phenoxyethanol  weighed.  singly  fish  The maximum  for one r e p l i c a t e ,  4  of sampling 4 f i s h (0.4  ml/1)  and  Each was k i l l e d by a sharp blow to the head and a  3 to 4 ml blood sample was taken immediately. was then s e p a r a t e d ,  The plasma  frozen and s t o r e d for l a t e r  The next 4 f i s h were s i n g l y a n e s t h e t i z e d then a d m i n i s t e r e d an o r a l dose of glucose weight).  In some  or fewer  were used for l e s s c r i t i c a l sampling times.  the  analysis.  and weighed,  (167 mg/100 g body  The glucose was d e l i v e r e d i n s o l u t i o n v i a  as d e s c r i b e d i n s e c t i o n  5.2.2,  treatment  (b).  syringe  The body  97  weight, glucose dose and time of for each f i s h . the  first  After recovery,  tank of the  series.  with the remaining f i s h .  the  The procedure was continued  except i n t u b a t i o n was mimicked  using a p i e c e of empty t u b i n g . 1  hours.  using a d i f f e r e n t  4 f i s h were placed in  The sham group was handled in the  same manner as the o t h e r s ,  approximately  i n t u b a t i o n were recorded  The e n t i r e  A second t r i a l  procedure took  was then  initiated,  chinook s t r a i n , with sampling times  staggered a c c o r d i n g l y .  R e p l i c a t e t r i a l s were c a r r i e d out on  separate  days,  1 week a p a r t .  5.1.1.5  Experimental Sampling  F i s h were captured from the glucose a d m i n i s t r a t i o n .  first  tank 3 hours  They were s i n g l y  after  anesthetized,  weighed and k i l l e d by a sharp blow to the head.  Blood was  drawn from each f i s h and handled as d e s c r i b e d in s e c t i o n 5.1.1.6.  Subsequent  samplings were c a r r i e d out on the  remaining groups at 6, respectively,  and the  12,  18,  24,  30 and 36 hours  sham group was sampled at  12.5  A f t e r every sampling, a c u r s o r y general h e a l t h was c a r r i e d out on each f i s h . in p a r t i c u l a r , were i n s p e c t e d infection 5.1.1.6 All  A l l major organs,  the  hours. check kidney  for l e s i o n s or other signs of  such as b a c t e r i a l kidney d i s e a s e  (BKD).  Blood Sampling Technique blood samples were taken from the caudal  a r t e r y / v e i n just posterior  to the a n a l f i n .  S e v e r a l mis of  98  blood were c o l l e c t e d  from each f i s h using a 22 gauge 1"  V a c u t a i n e r needle with a 4 ml V a c u t a i n e r c o l l e c t i o n c o n t a i n i n g sodium heparin a n t i c o a g u l a n t . on i c e no longer than 20 minutes, rpm for  p l a s t i c m i c r o c e n t r i f u g e tubes; i n s u l i n determinations.  Samples were kept  then c e n t r i f u g e d at  10 minutes to i s o l a t e the plasma.  sample was p i p e t t e d with a s t e r i l e  3,000  Each plasma  disposable  pipette  into 4  2 for glucose and 2 for  The plasma samples were immediately  frozen on dry i c e and s t o r e d at - 4 0 ° C for l a t e r 5.1.1.7  tube  analysis.  Plasma Glucose Determination  Plasma glucose was determined using a Glucose ( T r i n d e r ) Reagent K i t (Sigma K i t No. 315, L o u i s , MO, USA).  Frozen f i s h plasma was thawed at  F i v e ul of plasma were added to  temperature.  T r i n d e r reagent temperature.  and incubated for  The enzymatic  c o l o r with i n t e n s i t y proceeded as  2  2  2  St. ambient  1.0 ml Glucose  18 min at room  r e a c t i o n , which produced a pink  p r o p o r t i o n a l to glucose c o n c e n t r a t i o n ,  follows:  Glucose + H 0 + 0 H 0  Sigma D i a g n o s t i c s ,  2  - g l u c o s e oxidase-> G l u c o n i c a c i d + H 0 2  2  + 4-Aminoantipyrine + p-Hydroxybenzene Sulphonate  -peroxidases  Quinoneimine Dye + H 0 2  The absorbance of the s o l u t i o n was read i n a Shimadzu UV-160 Spectrophotometer calculated  at 505 nm.  Glucose c o n c e n t r a t i o n  from a standard curve prepared using  was  multi-levels  99  of glucose standards standards  set  (Glucose/Urea Nitrogen combined  C a t . No.  16-11, Sigma D i a g n o s t i c s ,  St.  Louis,  MO, USA). 5.1.1.8  Plasma I n s u l i n Radioimmunoassay (Conducted by D r . E . P l i s e t s k a y a , Dept. of Zoology, U n i v e r s i t y of Washington, S e a t t l e , WA, USA)  T h i s method was a m o d i f i c a t i o n  of F u r u i c h i  et  al.  (1980), Thorpe and Ince (1976) and T h o r e l l and Larsen (1978).  The f i r s t  stage of t h i s procedure  involved  p r e p a r a t i o n of u n l a b e l l e d and r a d i o l a b e l l e d anti-insulin  serum.  i n s u l i n , and  Salmon i n s u l i n was e x t r a c t e d  with a c i d -  acetone from f r e s h Langerhans i s l e t s and p u r i f i e d through a Sephadex G-50 column. dissolving phosphate  I n s u l i n standards  i n s u l i n in 0.1 buffer  were made by  N HCl and d i l u t e d with 0.04 M  to o b t a i n c o n c e n t r a t i o n s  of  0 to  MU/ml.  These were used to produce standard curves  et  1980).  al.,  iodinatation  1 2  5i  by the chloramine-T method  r a b b i t s with 200 jugrams of  salmon i n s u l i n  complete adjuvant  by a booster shot of  the l a s t  followed  in Freunds incomplete  interval.  Blood was sampled from the i n j e c t i o n and c e n t r i f u g e d  at  ml of v e r o n a l b u f f e r ,  injecting  in Freunds  adjuvant a f t e r  100 Mg of  a four week  r a b b i t s ten days 3,000 rpm for  Standard curves were produced by the 0.1  (Hunter  1962).  A n t i - s a l m o n i n s u l i n serum was obtained by  insulin  (Furuichi  R a d i o l a b e l l e d salmon i n s u l i n was prepared by  with  and Greenwood,  125  following  salmon i n s u l i n standards  after  15 mins. method: (0 -  125  100  MU / m l ) ,  '"i-salmon  were mixed t h r o u g h l y , total  radioactivity  i n s u l i n and a n t i - s a l m o n  i n s u l i n serum  incubated for 72 hours at 4°C and  (t)  was counted.  A total  of 2.5 ml of  80% ethanol was then added to each standard mixture by c e n t r i f u g a t i o n at 3,000 rpm for (ethanol  15 minutes at 4°C  causes the a n t i g e n - a n t i b o d y  precipitate).  followed  p r o t e i n complex  to  The supernatant was d i s c a r d e d and  r a d i o a c t i v i t y of the p r e c i p i t a t e  (b) counted.  r a t i o s were obtained from standards with low of u n l a b e l l e d i n s u l i n , because l i t t l e  High b : t concentrations  t r a c e r i n s u l i n was  i n h i b i t e d from b i n d i n g with the a n t i - s a l m o n  i n s u l i n serum,  which p r e c i p i t a t e d out of the mixture d u r i n g c e n t r i f u g a t i o n . Standard curves were drawn from the gamma counts  obtained  from each i n s u l i n c o n c e n t r a t i o n . In order to assay plasma samples, was repeated,  except that  0.1  ml of  the above procedure  f i s h plasma was added in  p l a c e of the  i n s u l i n standard.  Insulin concentration  extrapolated  from the standard curves  (Furuichi  et  was  al.,  1980). 5.1.1.9  Statistical  Analyses  Analyses were c a r r i e d out using the SAS a n a l y s i s variance (i)  (ANOVA) procedure (SAS v e r s i o n 6.03,  was a nested f a c t o r i a l experiment with  measures.  The experimental  1988).  of Part  repeated  f a c t o r s were s t r a i n and hour  chinook s t r a i n s x 9 sampling hours) and experimental replicates  were nested w i t h i n each s t r a i n and hour.  The  (3  101  i n d i v i d u a l measurements (tank)  represented  Cochran,  1980)  repeated measurements.  Disease i n the Part i i )  5.2.1  unit  (Snedecor and  Data from the B i g Qualicum chinook were not  i n c l u d e d i n the a n a l y s i s  5.2  taken w i t h i n an experimental  due to presence  of B a c t e r i a l Kidney  stock. O r a l Glucose Tolerance i n Chinook Salmon Acclimated to High and Low Carbohydrate D i e t s Respectively.  MATERIALS AND METHODS  5.2.1.1  Glucose Tolerance T r i a l s  T h i s s e r i e s of glucose t o l e r a n c e  t e s t s was conducted  using C a p i l a n o and Quinsam chinook s t o c k s .  T r i a l s were  c a r r i e d out between December 8 and 19,  1988,  and water  temperature ranged from 8 . 2 ° C to 9 ° C .  The C a p i l a n o and  Quinsam stocks were each d i v i d e d i n t o 2 groups diameter tanks) experiment  1.  ( i n two 8'  and a c c l i m a t e d to the 2 t e s t d i e t s used One group was fed the c o n t r o l  (low  carbohydrate) d i e t and the other was fed the  high  carbohydrate d i e t  for two weeks p r i o r to t e s t i n g .  glucose t o l e r a n c e  t r i a l s were conducted on each of the  in  Duplicate diet  groups of C a p i l a n o chinook, approximately one week a p a r t . The experimental p r o t o c o l and assay techniques study were s i m i l a r to those used i n p a r t ( i ) s e c t i o n s 5.2.3  through 5 . 2 . 7 ) .  used i n  (refer  to  this  102  Quinsam chinook had to be e l i m i n a t e d from the experiment due to the presence in the s t o c k , 5.2.1.2  of B a c t e r i a l Kidney  Disease  which became evident d u r i n g t o l e r a n c e  Statistical  testing.  Analyses  Analyses were c a r r i e d out using the SAS a n a l y s i s of variance (ii)  (ANOVA) procedure (SAS v e r s i o n 6.03,  was a nested  measures.  f a c t o r i a l experiment with  1988).  Part  repeated  The experimental f a c t o r s were d i e t and hour (2  a c c l i m a t i o n d i e t s x 9 sampling hours) and experimental replicates  were nested w i t h i n each s t r a i n and hour.  i n d i v i d u a l measurements (tank)  The  taken w i t h i n an experimental u n i t  represented repeated  measurements.  103 5.3  RESULTS  5.3.1  Part i ) Selected  Oral Glucose Tolerance i n Chinook Salmon of Strains  Significant differences tolerance  to the o r a l  t e s t between s t r a i n s c o u l d be d i s c u s s e d  terms of the o v e r a l l response points  i n response  curves.  distinguished  due to h i g h v a r i a t i o n and small sample  Plasma glucose and i n s u l i n l e v e l s of the groups were not s i g n i f i c a n t l y ( p r i o r to  different  12.5 hour sham  from r e s t i n g  levels  16 and F i g u r e 11 show plasma glucose and i n s u l i n  i n chinook salmon of three  strains,  before and a f t e r  a d m i n i s t r a t i o n of the o r a l glucose t o l e r a n c e Average body weight (± 1 s . d . ) 73 g i n H a r r i s o n chinook,  of the  215 ± 51.4  confounded with f i s h s t r a i n , and i t s a d j u s t e d for  in the a n a l y s e s  Plasma Glucose  In the a n a l y s i s glucose c h a l l e n g e ,  (see  test  g in Robertson Creek  effect  Body weight was c o u l d not be  discussion).  Response  of plasma glucose response  there was a s i g n i f i c a n t  to  between chinook  strains.  i n the response  oral  interaction  between s t r a i n and hour (p<0.05) i n plasma glucose indicating a difference  (GTT).  f i s h t e s t e d were 188 ±  chinook, and 263 ± 8 7 g i n N i t i n a t chinook.  5.3.1.1  sizes.  testing).  Table levels  only i n  Individual test  ( s t r a i n / h o u r means) c o u l d not be  statistically  glucose  levels,  p a t t e r n over  time  104  Table 16  - Mean plasma glucose and i n s u l i n c o n c e n t r a t i o n s in 3 s t r a i n s of chinook salmon subjected to an o r a l glucose t o l e r a n c e t e s t (GTT).  Strain  :  Harrison  Robertson C r .  Nitinat  Hour (post GTT) 76 + 5.4 2. 8 ± . 2 6  70 + 1 .9 4. 1 ± . 1 4  80 + 4.6<6)  78 + 7.5 2. 9 ± . 2 8  70 + 3.0 4. 2 ± . 3 2  72 + 1.8<6>  155 + 22.7< > 2. 4 ± . 1 2  157 + 10.6 4. 6 ± . 6 3  177 + 5.8< >  Glue Ins  252 + 21 .8 4. 4 ± . 5 0  244 + 9.8 5. i ± . 6 4  239 + 11.0  12  Glue Ins  308 + 20.0 4. 2 ± . 6 4  366 + 25.7 7. 1 ± . 4 0  385 + 15.0  18  Glue Ins  338 + 37.5 4. 6 ± . 6 2  436 + 22.4 6. 6 ± . 5 6  495 + 25.0  24  Glue Ins  269 + 17.3 4. 0 ± . 2 7  360 + 23.5 5. 9 ± . 6 2  402 + 38.7  30  Glue Ins  190 + 24.0 3. 6 ± . 6 0  328 + 34.8 5. 9 ± . 5 3  340 + 57.7  36  Glue Ins  188 + 25.9 4. 1 ± . 6 4  215 + 18.2 5. 9 ± . 4 9  263 + 30.9  180. 8 ± 2 9 . 5  295. 3 ±61 .6  389 .8 ± 5 0 . 1  0 (before)  Glue Ins  Sham (12.5)  Glue Ins  3  Glue Ins  6  Avg.  3  Weight ( g )  4  2  4  4  Plasma i n s u l i n was not assayed in the N i t i n a t s t r a i n . Mean plasma glucose c o n c e n t r a t i o n ± SEM (standard e r r o r of the mean). There was a s i g n i f i c a n t i n t e r a c t i o n between d i e t and s t r a i n in plasma glucose response (p<0.05) (see text for e x p l a n a t i o n ) . Mean plasma i n s u l i n c o n c e n t r a t i o n ± SEM. Plasma i n s u l i n response was s i g n i f i c a n t l y d i f f e r e n t between H a r r i s o n and Robertson Creek chinook (p<0.000l) (see text for e x p l a n a t i o n ) . Each plasma glucose and i n s u l i n value was obtained using 8 f i s h except where i n d i c a t e d in b r a c k e t s , beside glucose values. Average body weight of a l l f i s h used in t r i a l ± 1 standard deviation. 1  2  3  4  105  Figure 11: Oral Glucose Tolerance Test Response in Chinook Salmon of Selected B. C. Strains.  600  means ± 1 s.e.rrJ  H  1  1—  0 3 6 HARRISON  12 •  H  1  18  1  h  24  • ROB. CRK.  + 30 36 — - NITINAT H  H  c z  to  < 2  HOURS AFTER GLUCOSE ADMINISTRATION 1  Means ± 1 standard error of the mean, N=8.  106  Mean plasma glucose c o n c e n t r a t i o n s  ranged from 70 - 80  mg/dl i n chinook sampled j u s t p r i o r to the c h a l l e n g e sham groups sampled at  12.5 hours a f t e r mock  Plasma glucose c o n c e n t r a t i o n s challenge  peaked at  testing.  18 hours post  i n each of the three chinook s t r a i n s  peak mean of plasma glucose at the in H a r r i s o n chinook,  and in  tested.  The  18th hour was 338 mg/dl  436 mg/dl i n Robertson Creek chinook,  and 495 mg/dl i n N i t i n a t chinook.  In r e l a t i v e  terms,  the  glucose peak i n H a r r i s o n chinook was 77% that of Robertson Creek chinook and 68% that of N i t i n a t chinook. post glucose c h a l l e n g e , in the H a r r i s o n ,  concentrations  Robertson Creek and N i t i n a t chinook  remained s u b s t a n t i a l l y 263 mg/dl  mean plasma glucose  At 36 hours  elevated,  at  188 m g / d l , 215 mg/dl and  respectively.  Body weight and plasma glucose c o n c e n t r a t i o n , determined w i t h i n each sampling time correlated within 5.3.1.2  Response  Plasma i n s u l i n response  overall  were p o o r l y  strains.  Plasma I n s u l i n  was s i g n i f i c a n t  (hour),  over time  to the o r a l glucose  challenge  (p<0.05), and s i g n i f i c a n t l y  higher  i n Robertson Creek chinook than i n H a r r i s o n chinook  (p<0.000l).  Plasma i n s u l i n peaked at the  Robertson Creek chinook H a r r i s o n chinook indistinct  (see  concentrations  (4.6  (7.1  ng/ml),  F i g u r e 10).  12th hour in  ng/ml) and the  18th hour i n  although these peaks were very Resting plasma i n s u l i n  ( i n p r e - t r i a l and sham groups)  were a l s o  107  higher  in Robertson Creek chinook, averaging 4.15  compared to 2.85 for  ng/ml in H a r r i s o n chinook.  the N i t i n a t s t r a i n were not  ng/ml,  I n s u l i n data  available.  Body weight and plasma i n s u l i n c o n c e n t r a t i o n , w i t h i n each sampling time, strains.  as  computed  were p o o r l y c o r r e l a t e d w i t h i n  Poor c o r r e l a t i o n was a l s o found between plasma  glucose and i n s u l i n c o n c e n t r a t i o n ,  w i t h i n each  sampling  time. 5.3.2  Part i i ) E f f e c t of P r e - t e s t D i e t on O r a l Tolerance in C a p i l a n o Chinook Salmon  Glucose  Although t h i s study was conducted on both C a p i l a n o and Quinsam chinook s t o c k s ,  the l a t t e r  a n a l y s e s due to a high incidence  had to be e l i m i n a t e d  from  of B a c t e r i a l Kidney  Disease. Significant tolerance  differences  t e s t between d i e t  in response to the o r a l groups c o u l d be d i s c u s s e d  in terms of the o v e r a l l response c u r v e s . points  ( d i e t / h o u r means) c o u l d not be  statistically Table  glucose only  Individual test  distinguished  due to high v a r i a t i o n and small sample  sizes.  17 and F i g u r e 12 show plasma glucose and i n s u l i n  concentrations low ( c o n t r o l )  in C a p i l a n o chinook p r e - a c c l i m a t e d to or high carbohydrate d i e t ,  either  before and a f t e r  a d m i n i s t r a t i o n of the o r a l glucose t o l e r a n c e  test  (GTT).  108 Table  17  -  Mean plasma glucose and i n s u l i n c o n c e n t r a t i o n s in C a p i l a n o chinook salmon subjected to an o r a l glucose t o l e r a n c e t e s t (GTT) a f t e r feeding low or high carbohydrate p r e - t e s t d i e t s respectively. Diet  :  Low C a r b .  High C a r b .  1  1  Hour (post GTT) 65 ± 1.7(5) 5.6 +  68 ± 6 . 0 4.3 ± . 7 0  Glue Ins  61 ± 6.3(3) 3.9 ± . 4 7  61 ± 4 . 0 3 ) 5.0 ± . 2 7  3  Glue Ins  175 ± 18.6(3) 3.9 ±1 .05  165 ± 1 5 . 8 3 ) 4.5 ± . 9 1  6  Glue Ins  267 ± 5.6 10.5 ±1 .40  217 ± 31.9 5.8 ± 1 . 3 9  12  Glue Ins  388 ± 16.9 9.1 ± 1 . 1 0  302 ± 24.6 8.8 ± . 8 0  18  Glue Ins  464 ± 34.0 6.6 ± . 2 5  336 ± 30.8 9.2 ± . 5 1  24  Glue Ins  457 ± 3 7 . 9 7.4 ± . 3 8  30  Glue Ins  358 ± 34.7 5.3 ± . 4 8  206 ± 30.8 5.7 ± . 7 7  36  Glue Ins  250 ± 7.1<4) 4.6 ± . 5 6  211 ± 3 8 . 7 5.0 ± 1 . 0 1  4  399.7 ±  390.0 ± 69.9  0 (before)  Glue Ins  Sham (12.5)  Average weight ( g )  3  2  ( 4 )  75.9  ( 5 )  (  (  342 ± 3 3 . 1 7.6 ± 1 . 1 8  ( 4 )  ( 4 )  Low. ( c o n t r o l ) and high carbohydrate d i e t s from experiment 1 (see t a b l e 1 for c o m p o s i t i o n a l i n f o r m a t i o n ) . Mean plasma glucose c o n c e n t r a t i o n ± SEM (standard e r r o r of the mean). O v e r a l l plasma glucose response was s i g n i f i c a n t l y d i f f e r e n t between d i e t groups ( p < 0 . 0 0 l ) . Mean Plasma i n s u l i n c o n c e n t r a t i o n ± SEM. O v e r a l l plasma i n s u l i n response was not s i g n i f i c a n t l y d i f f e r e n t between d i e t groups (see text for e x p l a n a t i o n ) . Each plasma glucose and i n s u l i n value was obtained using 7 f i s h except where i n d i c a t e d i n brackets beside glucose values. Average body weight of a l l f i s h used i n t r i a l ± 1 standard deviation.  109  Figure 12: Effect of Pre-test Diet on Oral Glucose Tolerance Test Response in Capilano Chinook Salmon 600  E LU CO  o o  0  3  6  24  12  — LOW CARB. Diet  3 CO  36  • •• HIGH CARB. Diet  20  c  30  means ± 1 s.e.m.  15 +  10 +  z  5+  0  before  H  3  1  6  1  1  12  h  H  18  24  h  h—I  30  36  HOURS AFTER GLUCOSE ADMINISTRATION 1  Means ± 1 standard error of the mean, N=7.  110 5.3.2.1  Plasma Glucose  Response  Plasma glucose c o n c e n t r a t i o n s (p<0.001)  were s i g n i f i c a n t l y  in f i s h fed the low carbohydrate  (control)  than in those fed the high carbohydrate d i e t testing.  Plasma glucose peaked at  challenge  in both d i e t  groups,  mg/dl in c o n t r o l - a c c l i m a t e d  consistently (throughout  the  18 hours post glucose  In r e l a t i v e  terms,  464  the peak  of chinook a c c l i m a t e d to  was 72.5% that of c o n t r o l  Plasma glucose c o n c e n t r a t i o n s  lower  to  with peak means reaching  in plasma glucose c o n c e n t r a t i o n  acclimated f i s h .  prior  diet  f i s h and 336 mg/dl in high  carbohydrate-acclimated f i s h .  high carbohydrate d i e t  higher  the  dietwere  in the high carbohydrate d i e t  group  trial).  Plasma glucose was p o o r l y c o r r e l a t e d with body weight w i t h i n the 5.3.2.2  i n d i v i d u a l sampling times i n each d i e t  Plasma I n s u l i n  Response  Plasma i n s u l i n c o n c e n t r a t i o n s over time  groups.  concentration,  the  between d i e t  group at the  of  significantly  insulin  i n s u l i n response p a t t e r n s groups.  10.5  appear  Insulin concentrations  to peaked  6th hour, at a mean  n g / m l , and in the high carbohydrate  18th hour, at a mean l e v e l  Insulin concentrations  between  significant  and hour in plasma  in the c o n t r o l group at the concentration  significantly  Although there was no  i n t e r a c t i o n between d i e t  differ  changed  (p<0.05), but d i d not d i f f e r  the two d i e t  group.  of  9.2  ng/ml.  were s i m i l a r in the two d i e t  groups  Ill  at  12 hours post c h a l l e n g e ,  averaging  9.1  ng/ml and  ng/ml in c o n t r o l - and high c a r b o h y d r a t e - a c c l i m a t e d respectively.  N e v e r t h e l e s s these  were not s i g n i f i c a n t ,  possibly  data and small sample  sizes.  Poor c o r r e l a t i o n s  'apparent'  8.8 groups  differences  due to high v a r i a t i o n  in  the  were found between plasma i n s u l i n and  body weight, w i t h i n each d i e t group, at a given hour.  Plasma glucose and i n s u l i n c o n c e n t r a t i o n s  poorly  correlated.  sampling were a l s o  112 5.4  DISCUSSION  5.4.1  O r a l Glucose Oral  glucose  Tolerance:  tolerance testing  approaches because of  glucose  on  gastrointestinal  administration  insulin  (Cahill,  than  In t h i s d o s e of orally  167  mg  i n an  by  used  (1981,  1971). GTT  the  glucose  /  100  The  dosage used  1982a,  and  Poe  1982c).  delivered study, given  to the  glucose  fish  al.,  by way  i n these  was  of  trials  a  was  the  solution.  time  wall,  circulation.  to f i s h  yellowtail 100  capsules.  given  r e q u i r e d f o r enzymatic  delayed  the  Capsule  digestion  and  g / 60  kg  trials.  v i a c a p s u l e s e x h i b i t e d a lower i n comparison  same as  catfish,  r e v e a l e d i n the p r e l i m i n a r y t r i a l  plasma g l u c o s e The  in gelatin  calculated  delivered  bream and  i n human  been  stomach  (1987) i n c h a n n e l r e d sea  has  1978).  I t i s e q u i v a l e n t to the  orally  glucose  glucose  s t u d i e s mentioned p r e v i o u s l y , glucose  i t was  capsule  et  of  tolerance tests,  body w e i g h t d o s e commonly u s e d In t h e  indices  g body w e i g h t  Yone i n c a r p ,  of  intravenous  (Wolfe  solution  route  i t s effects  delivery  than  other  IV t e s t  glucose  and  other  Better correlation  and  80%  by W i l s o n  Furuichi  response  s e r i e s of g l u c o s e  intubation. that  with  chosen over  In humans, t h r o u g h  hormones, t h e o r a l  shown between t h e o r a l disposal,  was  i t i n v o l v e s a normal p h y s i o l o g i c a l  administration.  evokes a g r e a t e r  General  uptake of g l u c o s e  was In  that  this fish  elevation  the  sugar  in  breakdown of into  of  the  therefore introduced a  the  113  confounding f a c t o r  i n the t o l e r a n c e  testing.  The f a c t  that  glucose powder had to be a d m i n i s t e r e d in more than one capsule,  due to i t s  addition, capsules  b u l k , accentuated  the problem.  In  the wider tubing r e q u i r e d for o r a l d e l i v e r y of presented a greater  r i s k of trauma to f i s h than the  f i n e cannula tubing used for i n t u b a t i n g s o l u t i o n . reasons,  For these  the glucose s o l u t i o n - i n t u b a t i o n method was  A major c o n c e r n , however,  in using the  method was the p o s s i b i l i t y r e c o v e r i n g from anaesthesia response.  of  chosen.  solution-intubation  r e g u r g i t a t i o n , as  displayed a  fish  'cough-like'  I n t u b a t i o n of glucose s o l u t i o n c o n t a i n i n g a  b r i g h t food dye for v i s i b i l i t y  i n water d i d not  r e g u r g i t a t i o n w i t h i n the f i r s t  20 minutes post  During these t o l e r a n c e  trials  recovery.  temperature  ranged from 8.2  °C to  out on separate  days, approximately one week a p a r t ,  to e l i m i n a t e b i a s .  10 ° C .  seawater  reveal  R e p l i c a t e t r i a l s were c a r r i e d  Temperature must be taken i n t o  when a s s e s s i n g glucose t o l e r a n c e rate d e c l i n e s tolerance  with decreasing  would be expected  response.  temperature,  at lower  i n order account  As metabolic poorer glucose  temperatures.  Handling s t r e s s has been shown to cause hyperglycaemia in chinook salmon, which p e r s i s t s for many hours (Barton and Schreck,  1988;  S o i v i o and O i k a r i ,  1976).  Sham groups were  i n c l u d e d i n the GTT i n order to a s s e s s the e f f e c t procedure alone on blood g l u c o s e . 6 and 12 hours a f t e r and at the  mock treatment  of  the  Sham groups sampled at in a preliminary t e s t ,  12.5 hours i n a c t u a l t r i a l s ,  d i d not  exhibit  2,  114  e l e v a t i o n s i n plasma glucose c o n c e n t r a t i o n exceeding  100  mg/dl. 5.4.1.1  Part i ) O r a l Glucose Tolerance i n Chinook Salmon of Selected Strains  In t h i s different  examination of glucose t o l e r a n c e  strains,  addressed.  First,  there are two major p o i n t s  (see  known to be weight dependent,  exert  some e f f e c t  associated  that must be  the chinook s t r a i n s used i n these  were d i s s i m i l a r i n body weight is  i n chinook of  Table  Metabolism  and thus can be expected  on glucose t o l e r a n c e .  with chinook s t r a i n ,  16).  trials  it  As body weight  i s a confounding  to is  factor.  Due to the v a r i a b l e nature of the d a t a , and poor c o r r e l a t i o n s between body weight and plasma glucose within i n d i v i d u a l s t r a i n s , applied.  covariance analysis  In order to minimize the e f f e c t s of  was a d m i n i s t e r e d on a body weight b a s i s ,  because of the d i f f e r e n c e s strains,  conclusions  glucose t o l e r a n c e  c o u l d not be size,  therefore  chinook r e c e i v e d the same r e l a t i v e dosage.  regarding genetic differences  p o r t i o n of the glucose t o l e r a n c e  Second,  response  the  d i s p o s a l at each sampling time.  to e s t a b l i s h curves.  all  the in ascending,  curve expresses  the balance between the rate of glucose uptake and subsequent  glucose  Nevertheless,  i n mean body weight of  are not warranted.  level  It  glucose a b s o r p t i o n r a t e s from such  is  its difficult  response  115  The plasma glucose c o n c e n t r a t i o n s  in these chinook  t r i a l s are comparable to those found in rainbow t r o u t (Palmer and Ryman, tolerance.  and are i n d i c a t i v e  of poor glucose  Plasma glucose curves were s i m i l a r up to the  hour a f t e r  the glucose c h a l l e n g e ,  to d i v e r g e . challenge  1972)  at which time they began  Plasma glucose peaked at  i n each of the H a r r i s o n ,  N i t i n a t chinook s t r a i n s ,  reaching l e v e l s of  the challenge  plasma glucose e l e v a t i o n  18 hours post  (see  338 m g / d l ,  F i g u r e 11).  Continued  at 36 hours  reflected  after  some degree of  hyperglycaemia a s s o c i a t e d with s t r e s s and with the  fact  feed had been withdrawn for a p e r i o d of approximately hours at t h i s  response with i n d i s t i n c t  elicited  peaks of  a gradual  4.63  through the  declined gradually.  12th and 18th hours,  At the  36th hour, plasma  a l s o remained  ng/ml  respectively.  in plasma i n s u l i n appeared to commence at the  concentrations  100  insulin  ng/ml and 7.07  in H a r r i s o n and Robertson Creek chinook,  hour, continue  that  time.  The glucose challenge  rise  436  and remained e l e v a t e d at  in a l l s t r a i n s  glucose a d m i n i s t r a t i o n p o s s i b l y  glucose  Robertson Creek and  mg/dl and 495 m g / d l , r e s p e c t i v e l y , 36 hours a f t e r  6th  The  sixth  then  insulin  elevated.  R e s t i n g plasma i n s u l i n l e v e l s were in the range of 5 n g / m l , s i m i l a r to those found in other high in comparison to r e s t i n g relative  increase  concentration  fish species,  l e v e l s in humans.  in plasma i n s u l i n over  was l e s s than 2 - f o l d .  2 to but  The  fasting  By c o n t r a s t ,  humans  116  exhibit  8 to  10-fold  to 60 minutes a f t e r  increases  in plasma i n s u l i n w i t h i n 30  a glucose c h a l l e n g e  The magnitude and timing of the  (Cahill,  1971).  i n s u l i n peaks,  and poor  c o r r e l a t i o n s between plasma i n s u l i n and plasma glucose are c o n s i s t e n t with r e p o r t s by other glucose as a stimulant has b e e n ' d i s c u s s e d stimulus  investigators  regarding  for i n s u l i n r e l e a s e i n salmonids.  i n the L i t e r a t u r e Review,  for i n s u l i n s e c r e t i o n  in f i s h  As  the primary  i s plasma amino  acids. It  is  interesting  that  the plasma glucose response  the smaller H a r r i s o n chinook was lower than that of l a r g e r chinook of Robertson Creek and N i t i n a t especially  i n view of the  fact  i n d i c a t i v e of a s t r a i n d i f f e r e n c e  ruled out.  i n glucose u t i l i z a t i o n .  If plasma g l u c o s e . c o n c e n t r a t i o n s  enhanced i n s u l i n  response.  strains,  T h i s may be  in absorptive c a p a c i t i e s  lower i n H a r r i s o n chinook,  the  that carbohydrate metabolism  i s r e p o r t e d to increase with f i s h s i z e .  However, d i f f e r e n c e s  of  cannot be are  indeed  i t cannot be a t t r i b u t e d to an  117  5.4.2.1  Part i i ) E f f e c t of P r e - T e s t Diet on O r a l Glucose Tolerance  The a c c l i m a t i o n d i e t significant  effect  was demonstrated  to have a  on plasma glucose response.  the high carbohydrate d i e t  for two weeks p r i o r  a d m i n i s t r a t i o n of a glucose dose e x h i b i t e d plasma glucose c o n c e n t r a t i o n s fed the c o n t r o l d i e t .  Chinook  fed  to  27.5  than were evident  % lower peak in chinook  Plasma glucose peaked 342 mg/dl in  high c a r b o h y d r a t e - f e d chinook and at 434 mg/dl in c o n t r o l fed chinook,  with h i g h e s t l e v e l s observed at  post-glucose challenge.  This is  indicative  18-24  of an a d a p t a t i o n  response to the high carbohydrate d i e t ,  although  hyperglycaemia was s t i l l  persistent.  pronounced and  There appeared to be a d i f f e r e n c e response p a t t e r n s difference  in the two d i e t  is d i f f i c u l t  i n s u l i n responses observed concentrations chinook  in t h i s t r i a l  insulin  fold  in t h i s study.  had a higher average  i n s u l i n response to glucose c h a l l e n g e increase  Based on h i s  in  The C a p i l a n o body weight  Hilton postulated  t r o u t are s i m i l a r to t y p e - I I ,  deficiency factor  of  5 ng/ml).  represented  a 2-  concentration.  1987 study,  In other words,  the  insulin  390 g) and higher r e s t i n g plasma i n s u l i n l e v e l s Their  the  In comparison to  in part i ) ,  appear higher  in the  groups, although  to e v a l u a t e .  hours  that  rainbow  r a t h e r than t y p e - I ,  their diabetic-like  i n s u l i n , but r a t h e r ,  ( H i l t o n r e p o r t e d an i n c r e a s e  diabetics.  nature i s not due to a some other  inhibiting  in plasma i n s u l i n  in  118  fish  fed a high carbohydrate d i e t and concluded  that e l e v a t e d plasma glucose was not due to insulin).  therefore  insufficient  119 5.5  CONCLUSIONS  (Experiment 2)  Pronounced and p e r s i s t e n t hyperglycaemia o r a l glucose t o l e r a n c e tolerance  in chinook salmon of the  tolerance  examined.  initial  compared showed d i f f e r e n c e s  insulin concentration.  evident  Plasma  concentrations.  and there was a l s o a s t r a i n d i f f e r e n c e  circulating however  strains  f o l l o w i n g glucose a d m i n i s t r a t i o n  5 to 9 times over  three chinook s t r a i n s  to  t e s t i n g r e v e a l e d poor glucose  glucose c o n c e n t r a t i o n s increased  in response  The in glucose  in  No r e l a t i o n s h i p  between glucose t o l e r a n c e  was  and plasma  insulin  concentration. A c c l i m a t i o n to a high carbohydrate d i e t improvement decline  in glucose t o l e r a n c e ,  induced some  as i n d i c a t e d by a 30%  i n peak plasma glucose c o n c e n t r a t i o n s .  Poor  c o r r e l a t i o n between plasma glucose and i n s u l i n concentrations  indicates  secretagogue.  The improvement  demonstrated  that  glucose i s a poor i n glucose  in t h i s experiment  a d a p t a t i o n obtained  insulin  tolerance  supports the evidence of  i n Experiment 1,  in which f i s h  initially  responded to i m p o s i t i o n of a high carbohydrate d i e t r e d u c t i o n in growth r a t e ,  but subsequently  at a rate comparable to that of Although i t appears that differences  adapted and grew  fed the c o n t r o l  diet.  there may be some genetic  in glucose t o l e r a n c e ,  regarded as c o n c l u s i v e . a useful  fish  by a  the evidence should not be  O r a l glucose t o l e r a n c e  " s c r e e n i n g - t e s t " in examining glucose  Further r e s e a r c h i n c l u d i n g r a d i o t r a c e r work,  may serve as utilization.  enzyme  120  adaptation acclimation  and e f f e c t s of  longer  term carbohydrate  i n s e l e c t e d chinook s t r a i n s  is  recommended.  121 6  REFERENCES  Akiyama, T . , M u r a i , T . and T . 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